plant proteinaceous inhibitors of proteinases and a
TRANSCRIPT
PLANT PROTEINACEOUS INHIBITORS OF PROTEINASES AND a-AMYLASES
F. Garcia-Olmedo, G. Salcedo, R. Sanchez-Monge, L. Gómez, J". Royo and P. Carbonero
Departamento de Bioquímica. E.T.S. Ingenieros Agrónomos. Universidad Politécnica de Madrid. 28040 Madrid. Spain.
INTRODUCTION
P l a n t p r o t e i n s which a r e i n h i b i t o r y t o w a r d s v a r i o u s t y p e s of enzymes from a wide range of organisms have been extensively studied fo r m a n y y e a r s . P r o t e i n a s e i n h i b i t o r s h a v e r e c e i v e d p a r t i c u l a r a t t e n t i o n and a c c o r d i n g l y a n u m b e r of r e v i e w s conce rn ing t h e i r s t r u c t u r e , a c t i v i t y , e v o l u t i o n , p o s s i b l e p h y s i o l o g i c a l r o l e s and n u t r i t i o n a l p r o p e r t i e s h a v e a p p e a r e d r e g u l a r l y in t h e l i t e r a t u r e (Ryan, 1973, 1981, 1984; Laskowski and Kato, 1980; Richardson, 1981; Boisen, 1983). R e c e n t t e chn ica l advances in molecular biology have a c c e l e r a t e d the output of information about these inhibitors to the e x t e n t t h a t en t i r e ly new types have been uncovered and previously unsuspec ted re la t ionships have been es tab l i shed . These developments jus t i fy t h e p r e s e n t r ev iew t h a t will emphas ize the novel a spec t s , glossing over many important topics that have been adequately covered before. Among the most striking recent findings is the s t ructural and e v o l u t i o n a r y r e l a t i o n s h i p s of d i f f e r e n t c t - amylase i n h i b i t o r s wi th d i f f e r e n t t y p e s of p r o t e i n a s e i nh ib i t o r s , which is t h e r ea son for their joint consideration in this survey.
The i n h i b i t o r s have been g rouped for our p r e s e n t purposes as l i s t e d in Tab le 1, fol lowing an e c l e c t i c c r i t e r i o n : t h e f i r s t nine groups are t rue protein families, based on sequence homology, and the l a s t t w o a r e b a s e d on t h e m e c h a n i s t i c c l a s s e s of t h e e n z y m e s inhibi ted because not enough sequence information is available, and therefore may represent more than one protein family. A considerable n u m b e r of r e p o r t s d e a l w i t h i n h i b i t o r s t h a t h a v e n o t b e e n suf f ic ien t ly cha rac t e r i zed to discern whether they belong to any of the l is ted groups or represent new types. These cases have not been comprehensibly included in this review.
Table 1. Groups of protein inhibitors of proteinases and a-amylases
Inhibitor group Enzymes inhibited
2.
3.
4.
5.
6.
7.
8.
Soybean trypsin inhibitor (Kunitz) family
Bowman-Birk inhibitor family
Cereal trypsin/a-amylase inhibitor family
Potato inhibitor I family
Potato inhibitor II family
Squash inhibitor family
Barley protein Z/a-,-ant i trypsin family
Ragi I-2/maize bifunctional inhibitors family
9. Carboxypeptldase A, B inhibitor family
10. Thiol-proteinase inhibitors
11 . Cathepsin D and Pepsin i n h i b i t o r s
Serine proteinases and endogenous a-amylases
Serine proteinases
Serine proteinases and heterologous a-amylases
Serine proteinases
Serine proteinases
Serine proteinases
Serine proteinases
Serine proteinases and heterologous a-amylases
Metal lo-carboxypeptidases
Endogenous and heterologous th io l -p ro te inases
Carboxyl-protei nases
All the serine proteinase inhibitors treated here seem to obey a standard mechanism, which has been thoroughly studied from different points of view (see Laskowski and Kato, 1980), including the detailed e l u c i d a t i o n by X-ray c rys t a l log raphy of the th ree-d imens iona l structure of several enzyme-inhibitor complexes, as reviewed recently by Read and James (1986). In summary, the inhibitors are highly specific substrates for their target enzymes, which undergo a limited and extremely slow proteolysis, so that the system behaves as if the free enzyme and the inhibitor were in simple equilibrium with the enzyme/inhibitor complex. On the surface of each inhibitor there is at least one reactive bond (Pl-P'l) which interacts with the active s i te of the enzyme. An inhibitor molecule which has undergone hydrolysis of i ts r eac t ive bond is as act ive as the unhydrolised inhibitor and is able to form a stable complex with the enzyme. In most, but not all , of these inhibitors, the react ive site peptide bond is encompassed in at least one disulphide loop, which ensures that during conversión of the original to the modified inhibitor the two peptide chains cannot dissociate. The nature of the amino acid residue at the carboxyl side, P l position, generally determines the proteinase inhibited: Lys or Arg for trypsin-like enzymes; Phe, Tyr, or Leu for chymotrypsin; and Ala for elastase. The amino side of the peptide bond, the P' l position, does not seem to be involved in determining specificity (Laskowski and Kato, 1980).
More than one react ive site is sometimes present in a single polypeptide chain, in which case more than one enzyme molecule of the same or different specificity can be simultaneously inhibited by a single inhibi tor molecu le . In some of these cases , it is fairly obvious that the múltiple reactive sites are associated with múltiple protein domains that must have been generated from an ancestral domain by i n t e r n a l gene dupl ica t ions , while in o ther cases a convergent evolutionary process cannot be excluded.
In subsequen t p a g e s , we will f i rs t deal with the d i f fe ren t inh ib i to r groups , and then , the i r possible impl ica t ion in plant metabolism, plant protection, and human and animal nutrition will be addressed.
SOYBEAN TRYPSIN INHIBITOR (KUNITZ) FAMILY
The crystallization of a trypsin inhibitor from soybean (STI) and of its complex with trypsin, which was carried out over forty years ago by M. Kunitz (1945, 1946, 1947a,b, 1949), was one of the major achievements in the early stages of research on protein inhibitors from p lan t s . Fur ther studies concerning its amino acid sequence (Koide and Ikenaka , 1973a,b ; Koide e t a l . , 1973), i t s t h r e e -dimensional s t ruc ture (Sweet et al . , 1974), and its mechanism of interaction with the enzyme (Sealock and Laskowski, 1969; Kowalski et al., 1974; Kowalski and Laskowski, 1976a,b; Hunkapiller et al., 1979; Baillargeon et al., 1980) made STI the first plant inhibitor to be well charac ter ized . In spite of this early s tar t , the identification of further members of the STI family in other species has been a rather slow process that has recently acquired momentum with the identification of a number of new inhibitors and, especially, with t h e r e a l i z a t i o n t h a t t h e i n h i b i t o r s of s u b t i l i s i n / e n d o g e n o u s a-amylase from cereals are indeed homologues of STI (Hejgaard et al., 1983; Svendsen et al., 1986; Maeda, 1986).
Distribution and Inhibitory *Properties
Inhibi tory p ro t e in s with Mr of about 20,000, two disulphide bridges and similar amino acid composition to that of STI have been identified in a wide range of species. Although STI appears as a single form for which two addi t ional al íeles have been found, mixtures of numerous isoforms with different inhibitory properties are present in many species (Table 2). A majority of the identified inhibi tors a re specif ic for e i ther trypsin or chymotrypsin, with either weak or nuil activity for the second enzyme, while a few are about equally effective against both enzymes. Some members of this family are only weak inhibitors and it can be speculated that they might be active against other, unidentified enzymes. Only one of
Table 2. Distribution and inhibitory properties of members of the STI (Kunitz) family
Species Inhibitor Specificity* Inh/Enz Selected references
Papilionideae
Psophocarpus tetrágono!obús WTI1A,IB,2,3 T-s 1:1 Yamamoto et al.1983 (winged bean) WTCI1 T-s, Ch-s 1:1 Shibata et al. 1986
WCI1 Ch-s 1:1 WCI2,3 Ch-s 1:2 WCI4 Ch-w
Erythrina latissima DE1 Ch-s Joubert et al. 1985 DE3 T-s 1:1
E. cristagalli DE1.8 T-s, Ch-w 1:1 Joubert and Sharon, DE2,4 Ch-s 1:1 1985 DE3 Ch-s 1:2
E. corol ladendron DE1,5 Ch-s 1:1 DE6,7 T - s , Ch-w 1:1 DE8 T-s 1:1
E. acanthocarpa DE1 T -s , Ch-w DE2 Ch-s
E. caffra DE1 T - s , Ch-s DE2 Ch-s DE3 T - s , Ch-w DE4 T-s
E. humeana DE3 T -s , Ch-w
E. lysistemon DE1 Ch-s DE2-4 T -s , Ch-w
E. seyheri DE1,3,5 T -s , Ch-w DE2.4 Ch-s
Glycine máxima STIa,b,c T-s 1:1 Kim et a l . 1985
these inhibitors, DE3 from Erythrina latissima, has also been shown to inhibit tissue plasminogen activator (Joubert et al., 1985). The cereal inhibitors are isoform mixtures active against subtilisin and endogenous a-amylases (War chale wski, 1977a,b; Yoshikawa et al., 1976;
Table 2. Continued
Species Inhibitor Spedfici ty* Inh/Enz Selected references
Cesalpinoideae
Peltophorum africanum DE1 T-s Joubert 1981
Mimosideae
Adenanthera pavonina (caro!i na tree)
DE1-8 T-s, Ch-s (DE5)1:1 Richardson et al. 1986
Sudhakar Prabhu and Pattabiranam 1980
Albizzia julibrissin (silk tree)
AII.III BI.II
T-s, Ch-s Ch-s
1:1 Odani et al. 1979
Acacia elata T-s Kortt and Jermyn 1981
A. sieberana T-s, Ch-s 1:1 Joubert 1983
Gramineae
Hordeum vulgare eum yuigt (barley}
BASI S-s, aA-s 1:1+1 Svendsen et al.1986
Triticum aestivum (wheat)
WASI S-s, aA-s 1:1+1 Mundy et al. 1984 Maeda 1986
Sécale cereale (rye)
RAS I S-s, aA-s Weselake et al. 1985
Mosolov and Shulgin 1986
*T , t r y p s i n ; Ch, chymotrypsin; S, s u b t i l i s i n ; aA, a-amylase; s , s t rong ; w, weak
Weselake et al., 1983a,b; Mundy et al., 1983; Hejgaard et al., 1983). More specifieally, the a-amylase 2 isozymes from barley, wheat, rye and oats are inhibited, while the a-amylase 1 isozymes are not (Mundy e t a l . , 1984). Amylases from sorghum, r i ce , sa l iva , pánc reas , Aspergillus oryzae or Bacillus subtilis were found to be insensitive (Mundy et al., 1984). Not included in Table 2 are certain inhibitors whose c l a s s i f i ca t ion as Kuni tz - type is s t i l l uncer ta in , although their Mrs and amino acid compositions are compatible with such a classification. This is the case for the trypsin inhibitor CPPTI-fm
from Cucúrbita pepo (Pham et al., 1985), the kallikrein inhibitors from p o t a t o e s (Hojima et al . , 1973), a protease inhibitor from spinach leaves (Satoh et al., 1985), a trypsin inhibitor from white mustard (Menegatti et al., 1985), and the trypsin inhibitors from the tube r s of t a r o (Ogata and Makisumi, 1984). The l a t t e r are of p a r t i c u l a r i n t e r e s t because their dimeric nature has been well established (Ogata and Makisumi, 1985).
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The in te rac t ion between the Kuni tz- type inhibitors and those enzymes agains t which they show strong inhibitíon is generally s t o i c h i o m e t r i c , as summarized in Table 2. All repor ted trypsin inhibitors form a 1:1 complex with the enzyme and those few that are also effective against chymotrypsin form a 1:1 complex with this enzyme, which can be displaced from the complex by trypsin. Most of the inhibitors that only inhibit chymotrypsin form a 1:1 complex, but certain inhibitors from Psophoearpus and Erythrina are able to form 1:2 complexes . In th i s con tex t , it is of in te res t to note tha t Bosterling and Quast (1981) demonstrated the ability of STI to bínd two molecules of chymotrypsin, an enzyme that is not inhibited by it, while only one molecule of chymotrypsin was bound if the inhibitor had been previously incubated with trypsin. The cereal inhibitors are able to s imultaneously bind one molecule of subtilisin and one molecule of a-amylase (Mundy et al., 1983, 1984; Weselake et al., 1983a,b; Halayko et al., 1986).
The inhibitors from species of the Mimosidae subfamily, such as Acacia, Albizzia, and Adenanthera spp. , a re composed of two disulphide- linked chains, whereas the remaining inhibitors from Leguminoseae and those from Triticeae are single-chained. It seems likely, from their alignment with the single-chain inhibitors, that the two chains are originated from a single chain precursor (see Fig. 1).
Figure.l. (opposite) Alignment of amino acid sequences of members of the soybean trypsin inhibitor (Kunitz) family. WBI is a trypsin inhibitor from winged bean, Psophoearpus tetragonolobus (Yamamoto et al., 1983); ELI is trypsin inhibitor DE3 from Ery thr ina l a t i s s i m a (Joubert et al., 1985); STI is the Kunitz trypsin inhibitor from soybean (Kim et al., 1985); PAI is a trypsin inhibitor from Peltophorum africanum (Joubert, 1981); AJI1 and AJI2 are partial sequences of the two-chained inhibitors AII and BU from Albizzia julibrissin (Odani et al., 1979); API is the two-chained trypsin inhibitor DES from Adenanthera pavonina (Richardson et al., 1986); AEI and ASI are partial sequences of the two-chained trypsin inhibitors from Acacia elata (Kortt and Jermyn, 1981) and A*cacia sieberana (Joubert, 1983), respeetively; BASI and WASI are inhibitors of subtilisin and of endogenous oc-amylases from barley (Svendsen et al., 1986) and wheat (Maeda, 1986), respeetively; RPI is a rice inhibitor (Kato et al., 1972; cited by Mundy et al., 1984). Vertical arrows (1) indícate the reactive bonds of the trypsin inhibitors. Gaps introduced for the alignment are indicated (-): The N-terminal positions of the second chain of the two-chained inhibitors are indicated by a vertical Une (\). Conserved positions are boxed. Unidentified residues are indicated by an asterisk (*).
Structure and Evolution
Avai lab le sequenee da t a for homologues of STI (Kunitz) in different taxa are summarized in Fig. 1, and the homology matrix derived from it is presented in Table 3, where only common segments of the sequences were used to calcúlate percent homology in the cases in which the sequences were i ncomple t e . As expec ted , higher homologies are observed within each of the Leguminosae sub-families and among the cereal inhibitors, than between these taxonomic groups, with the n o t a b l e e x c e p t i o n of STI i tself , which seems to be significantly closer to the inhibitor from Peltophorum (sub-family Caesalpinoideae) than to those of Erythrina and Psophocarpus, which belong to the Pap i l ionaceae sub-family , t oge the r with Glycine (soybean). Inhibitors from the most primitive of the Leguminoseae sub-families, Mimosideae, are closer to that of Peltophorum and to STI than to the other two inhibitors from the Papilionideae. The c e r e a l i n h i b i t o r s a r e s i g n i f i c a n t l y c lose r to those from the Mimos ideae than to those from the o the r two Leguminoseae sub-families. A complete sequenee of the Peltophorum inhibitor, as wel l as f u r t h e r s e q u e n c e s from the Caesa lp ino ideae and t h e Papilionideae, should help to clarify the evolutionary relationships suggested by the present data.
Table 3. Binary comparisons (% homology) o f i nh ib i t o r s f rom the STI (Kuni tz) f am i l y . Homology was ca lcu la ted f o r common segments of the sequences tha t appear i n F i g . 1 . Abrev ia t ions are as in F ig . 1 .
PAPILIONIDEAE CESALPINOIDEAE MIMOSIDEAE GRAMINEAE » — i •
i 1 | , , ,
NHIBITOR
WBI
ELI
STI
PA1
AJI1
AJI2
API
AEI
ASI
BASI
WAS I
RPI
WBI ELI
66
•
STI
45
42
PAI
48
45
67
AJI1
40
33
51
50
AJI2
46
39
52
48
59
API
34
34
39
52
68
68
AEI
37
33
43
45
79
58
61
ASI
41
37
37
53
53
54
49
65
BASI
28
26
30
33
32
37
26
32
36
WAS I
26
26
30
38
32
42
26
36
36
89
RPI
41
33
41
43
30
43
41
27
30
67
67
The reactive bond has been identified in five of the Leguminoseae inhibitors as Arg-Ile or Arg-Ser in exactly homologous positions (Fig. 1). Since no chymotrypsin inhibitor of this group has been sequenced, the apparent conservation of Arg at the P l position probably ref lec ts this bias. The inhibitors from wheat and barley, which do not inhibit trypsin, respectively have Gly-Ala and Val-Ala in the homologous positions. High variability for the reactive site amino acids have been recognised since early times in proteinase inhibitor research (see Laskowski and Kato, 1980). More recently, this observation has been extended to those amino acids which are in contact with the enzyme, in the case of the ovomucoids (Laskowski et al., 1987), and to a domain of 16 amino acids around the reactive site, in the case of the serpins (Hill and Hastie, 1987). An X-ray c r y s t a l l o g r a p h i c ana lys i s of the STIa-porc ine t rypsin complex indicated that STI was in contact with the enzyme at the following p o s i t i o n s ( s e e F i g . 1 ) : A s p - P h e ( 4 - 5 ) A s n ( 1 6 ) , Pro-Tyr-Arg-Ile-Arg-Phe (65-70), His-Pro (75- 76). Data in Fig.l are compatible with con tac t área hypervariabi l i ty for this group of inhibitors, with the exeeption of Asn (16), which seems to be fairly conserved, and the already mentioned Arg at the Pl position.
A search for internal repeats was undertaken following the reports of two binding sites for chymotrypsin in STI (Bosterling and Quast, 1981), in WCI2,3 from winged bean (Shibata et al., 1986), and in DE3 from Erythrina cristagalli ( Jouber t and Sharon, 1985). Mostly imperfect short repeats were found and their distribution was not consistent with a straight-forward two-domain structure. Perhaps when inhibitors with a 1:2 stoichiometry are sequenced, a clearer picture will emerge.
A crystallographic study of wheat WASI is in progress (Maeda et al . , 1987) and an elucidation of its react ive site with subtilisin, as well as of the contact áreas with both subtilisin and endogenous a-amylase would be of great interest. The 3.0 A electrón density map is in agreement with predictions of secondary structure based on the amino acid sequence and indicates a similar structure to that of STI (Maeda, private communication). In this context, a survey of the act ivi ty against endogenous ot-amylases of the inhibitors from the Leguminoseae would help to clarify whether this activity was either lost during the evolut ion of the l ipid-storing species , acquired du r ing t h e e v o l u t i o n of spec i e s with s t a r c h y seeds , or s t i l l conserved in at least some «members of both branches.
Genetics
Gene t i c v a r i a n t s of STI, designated a, b, and c, have been iden t i f i ed , a f t e r sc reen ing the USDA germplasm co l lec t ion by polyaerylamide gel electrophoresis , and found to be allelic forms encoded at a single locus for which a nuil alíele also exists (see Orf and Hymowitz, 1979). Isolation and charac te r iza t ion of the genetic variants was carried out (Freed and Ryan, 1978; Kim et al., 1985): the most frequent a l íe le , STIa (88.8%), differs from the
second, STIb (10.9%), at 8 sequence positions, whereas the less frequent one, STIc (0.3%), differs at only one position from STIb, which has led to the speculation that differentiation of the first two alíeles was quite ancient and had already been completed in Glycine soja, the wild progenitor of the cultivated species Glycine max (Kim et al., 1985). The STI locus has been assigned to linkage group 9 in G. max, at 16.2±1.5 map units from the acid phosphatase locus (Hildebrand e t a l . , 1980) and at 15.3±0.9 of the leucine aminopeptidase locus (Kiang and Chiang, 1986).
The gene encoding the barley inhibitor has been assigned to barley chromosome 2, a f te r immunochemical analysis with monospecific an t ibodies of al l ba r l ey -whea t addi t ion l ines (Hejgaard et a l . , 1984a), and that for the rye gene has been similarly assigned to chromosome 2R (Hejgaard et al., 1984b).
Physiology
Messenger RNA for STI was isolated and translated in vi tro by Vodkin (1981). A polypeptide was obtained that had higher apparent Mr than that of the inhibitor obtained from mature seeds, Mr 23,200 vs. Mr 21,500. This finding was confirmed by sequencing a cDNA clone which encoded a protein with a putative leader sequence of 25 a mino acids preceding the known sequence of STI (Hoffman et al., 1984). The ultrastructural localization of STI in thin sections was carried out by Horisberger and Tacchini (1982), using specific antibodies and protein-A-gold label. In the cotyledons, STI was found in the cell wall and in most of the protein bodies, but not in the cytoplasm, while in the embryonic axis, so me of the label was also associated with the cytoplasm. An investigation of the fate of STI during germination has shown a rather precise proteolytic processing at the carboxyl terminus, which seems to involve a five residue sequence in STIa (Hartl et al., 1986). This would argüe against its possible role as a store for sulphur a mino acids and would suggest so me unknown specific function in germination.
The interaction of barley a-amylase II with BASI has been studied in detai l by eLectrophoresis and by a variety of physical methods (Waselake et al., 1983a; Mundy et al., 1983; Halayko et al., 1986). The inhibi tor combines with the enzyme at a molar ra t io 2 :1 , a f f ec t ing an enzymic tryptophan residue which is essent ia l for p roduc t ive enzyme-subs t ra te binding. The inhibitor seems to be synthesized in barley endosperm at least up to 30 days after anthesis and its mRNA is not detected in mature dry aleurone, although it appears upon "in vitró" incubation and is dramatically induced by t reatment with abcisic acid (Mundy and Rogers, 1986). In a study where de-embryonated seeds were incubated with labelled a mino acids, the enzyme-inhibitor complex was not significantly labelled (Rodaway, 1978), suggesting a possible regulatory role of the inhibitor during seed development, by inhibiting the enzyme during starch synthesis, and/or, in the event of premature sprouting, by preventing starch degradation (Mundy et al., 1983).
BOWMAN-BIRK INHIBITOR FAMILY
Following the discovery of antitryptic activity in soybeans, two d i f f e r e n t i n h i b i t o r s were e v e n t u a l l y i d e n t i f i e d , t he a l r e a d y deseribed Kunitz type and a second one, separated by Bowman (1946). A characterization of the second inhibitor was carried out by Birk et al. (1963a,b), henee its present designation as Bowman-Birk inhibitor (BBI), and its covalent s t ruc ture was establ ished by Odani and Ikenaka (1972, 1973a). Homologues of BBI have been isolated in the seeds of numerous species. A first-hand account of the early research on BBI has been published by Birk (1985) and a thorough review of the BBI family has appeared quite recently (Ikenaka and Norioka, 1986). A succinct description of this family will be presented here and only very recent developments will be treated in some detail.
Distribution and Inhibitory Properties
The Bowman-Birk inhibitor (BBI) is a protein of 71 amino acid r e s i d u e s , wi th 7 d isulphide b r idges , t h a t inh ib i t s two se r ine proteases simultaneously, trypsin and chymotrypsin. The double-headed nature of this inhibitor was elegantly corroborated by Odani and Ikenaka (1973b), who fragmented the molecule with cyanogen bromide and pepsin into the two active domains and a C-terminal tetrapeptide. Homologues of BBI, with two reactive sites and capable of binding two molecules of serine proteases simultaneously, have been deseribed in a number of species. In general, specificity is determined by the residue at Pl position of the reactive site: arginine and lysine for trypsin, tyrosine and phenylalanine for chymotrypsin, and alanine for elastase (see Ikenaka and Norioka, 1986, and Fig. 2). Exceptions are the f i r s t r e a c t i v e s i t e s of the peanut inhib i tors (Norioka and Ikenaka, 1983b, 1984) and the second reactive site of the soybean inhibitor C- II (Odani and Ikenaka, 1977), which can bind either trypsin or chymotrypsin, although an Arg residue at the Pl position would predict specificity for trypsin in all the cases. A different exception is that represented by inhibitor II from azuki bean, which can not bind trypsin and chymotrypsin simultaneously, although it is truly double-headed (Yoshikawa and Ogura, 1978).
Inhibitors of the Bowman-Birk type have been deseribed in the seeds of many Leguminoseae: soybean (Odani and Ikenaka, 1972, 1976, 1977), garden bean (Wilson and Laskowski, 1975), azuki bean (Ishikawa et al., 1979; Yoshikawa et al., 1979a; Kiyohara et al., 1981), mung bean (Zhang et al., 1982; Wilson- and Chen, 1983), cowpea (Morphy et al . , 1985), lima bean (Stevens et al., 1974), Macrotyloma axillare ( Joube r t e t a l . , 1979), Lonchocarpus capassa ( Jouber t , 1984a), Pterocarpus angolensis (Joubert, 1982), Vicia angustifolia (Shimokawa et a l . , 1984), peanut (Norioka and Ikenaka, 1983a,b), navy bean (Wagner and Riehm, 1967), kidney bean (Putzai, 1968), black-eyed pea (Gennis and Cantor, 1976), runner bean (Hory and Weder, 1976) and lentil (Weder, 1986). Until recently, the BBI family was thought to
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Table 4. Binary comparisons (% homology) of members of the Bowman-Birk inhibitor family. Homology was calculated for common segments of the sequences that appear 1n Fig.2. Abreviations for inhibitors are as in Fig. 2. Tribes were abreviated as follows: Gly, Glycineae;Pha, Phaseoieae; Vic, Vicieae; Sti ,Stj^ losantheae; Dal, Dalberg1ae;Gra, Gramineae.
GROUP - — - I II III IV
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Gly SBI1 77 72 73 75 72 75 67 65 63 63 62 64 47 38 35 41 66 39 50 31 31
Gly SBI2 69 72 75 69 69 67 59 59 61 60 61 49 38 35 39 63 36 50 35 33
Pha GBI 77 78 74 79 57 64 69 65 65 67 44 42 40 41 66 37 61 37 33
Pha ABI1 97 79 83 62 67 69 64 66 65 48 39 35 39 61 36 61 35 35
Pha ABI2 82 88 63 69 71 66 68 67 50 41 38 41 65 38 61 35 35
Pha M8I1 93 63 67 68 68 67 67 45 35 33 36 64 37 58 31 33
Pha MBI2 61 70 69 67 68 68 46 37 32 37 64 37 65 35 35
Gly BBI 75 79 75 75 74 45 34 34 36 65 39 61 37 41
Pha BTCI 83 76 79 67 44 35 30 37 63 38 61 38 35
Pha LBI 77 81 72 44 38 33 41 59 40 65 38 39
Pha ABI3 74 63 43 35 31 37 59 37 58 37 37
Pha MAI1 68 48 37 33 39 67 39 65 37 37
Pha MAI2 46 35 31 36 58 33 69 35 39
III | V1c VAI 43 43 43 42 40 54 36 33
Sti PI1 87 75 37 37 36 27 27
Sti PI2 75 31 34 32 27 27
Sti PI3 37 46 35 29 31
Dal LCI 35 54 33 33
Dal PAI 33 27 31
Vic CPI 54 58
Gra WGI1 66
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Figure 2. (jopposite). Alignment of a mino acid sequences oí proteinase inhibitors of the Bowman-Birk family. SBI1 and SBI2 are respectively inhibitors CII and DII from soybean (Odani and Ikenaka, 1976, 19 77); GBI is inhibítor IV from garden bean (Wilson and Laskowski, 1975); A BU and ABI2 are inhibitors I- A, A' and IIa,c, respectively, from azuki bean (Kiyohara et al., 1981; Ishikawa et al., 1985); MBI1 and MBI2 are two inhibitors from mung bean (Zhang et al., 1982; Wil son and Chen, 1983); BBI is the Bowman-Birk inhibitor from soybean (Odani and Ikenaka, 1972); BTCI is an inhibitor from cowpea (Morphy et al., 1985); LBI is inhibitor IV-IV from lima bean (Stevens et al., 1974); ABI3 is inhibitor I-II from azuki bean (Ishikawa et al., 1979; Yoshikawa et al., 1979a); MAI1 and MAI2 are respectively inhibitors DE3 and DE4 of Macrotyloma axillare (Joubert et al., 1979); VAI is an inhibitor from Vicia angustifolia (Shimokawa et al. , 1984); PI1, PI2 and PI3 are respectively inhibitors AII, Bill, and BU from peanuts (Norioka and Ikenaka, 1983a,b); LCI is inhibitor DE4 from Lonchocarpus capassa (Joubert, 1984a); PAI is inhibitor DE1 from Pterocarpus angolensis (Joubert, 1982); CPI is an inhibitor from chickpea (Belew and Eaker, 1976); WGI1 and WGI2 are inhibitors from wheat germ (Odani et al., 1986). Vertical arrows (i) indícate the reactive bonds. Gaps are indica ted (-)
Structure and Evolution
Inhibitors of the BBI family have been classified into four groups according to the ir sequence homology and a phylogenetic tree has also been deduced (see Ikenaka and Norioka, 1986). In Fig. 2 the sequences included in the alluded elassification have been aligned with the following additional ones: cowpea inhibitor (Morphy et al., 1985), chickpea inhibitor (Belew and Eaker, 1976), two sequences from spec ies of the t r ibe Dalbergieae , namely Lonchocarpus capassa (Joubert, 1984a) and Pterocarpus angolensis (Joubert, 1982), and two t ryps in i n h i b i t o r s from wheat germ (Odani e t a l . , 1986). The percentage of homology for the length of sequence compared in each case is presented for all binary comparisons in Table 4. From these data the following aspects merit attention:-
i) Although quite diverged, the cereal inhibitors are more closely related to each other than to any other of the inhibitors. ii) The inhibitor from chick pea is about equidistant to the cereal inhibitors (54%-58% homology), to group I (5096-65%), to group II (61%~69%), and to the only other sequenced inhibitor from the tribe Vicieae (Group III, 54%). iii) The inhibitor from cowpea fits into group II. iv) The two inhibitors from the tr ibe Dalbergieae are quite different from each other (35% homology); while LCI-DE4 is cióse and about equidistant to group I (61%-66%) and to group II (58%-67%), PAI-DE1 is qu i te apar t from all the other inhibitors (33%-46%). v) Inhibitors from the same species, i.e. soybean, belong to more than one group.
These observat ions clearly indicate that a good par t of the divergence of the multigene families encoding inhibitors of the BBI-type occurred before speciation of the Leguminoseae and that the apparent lack of correlat ion between the sequence data and the taxonomie elassification is due to non-homology of the loci being compared, which makes the above-mentioned phylogenetic tree difficult to in terpret in evolutive te rms. Besides sequencing new inhibitors from new species, a clear priority from an evolutionary point of view would be to de te rmine all the different sequences of BBI-type inhibitors within a number of species and to investígate the genome organization of tpe corresponding multi-gene families.
An ancestral monovalent inhibitor that would have generated the double-headed ones by internal gene duplication has been postulated (Tan and Stevens, 1971; Odani and Ikenaka, 1976). In this context, the recent finding of single-headed inhibitors in wheat germ further suppor ts the hypothes is and suggests t ha t these are the most primitive among the knbwn members of this family. Then, the cereal double-headed inhib i tors would be the closest to the ances t ra l d o u b l e - h e a d e d i n h i b i t o r , b e i n g t h e l e s s d i v e r g e d from t h e single-headed one, and the inhibitor from chickpea would be the most primitive among the dicot inhibitors, being the closest to the wheat ones. However, the single-domain cereal inhibitors could be the result of post-translational processing of double-headed precursor or could be encoded by genes originated through deletion of the second
domain sequenee. Molecular cloning of the pertinent genes and further p r o t e i n s e q u e n e e s might allow to d i s c r i m í n a t e be tween these alternatives.
Very recently, the structure of the BBI-type inhibitors AII from peanut has been determined by X-ray crystallography at a 3.3 A resolution (Suzuki et al., 1987). The structures of the two domains are similar and are related by an intramolecular approximate twofold r o t a t i o n axis, while the two independent pro tease binding s i tes protrude from the molecular body on opposite sides. Based on this three-dimensional s t ruc ture , the evolutionary scheme proposed by Odani and Ikenaka (1976) has been further refined (Suzuki et al., 1987), postula t ing a second par t i a l duplication, that would have extended the C- terminal and in turn replaced the N-terminal residues of the first domain, and a change of the pairing partners of the disulphide bridges, as represented in Fig. 3.
Other r e c e n t s t r u c t u r a l s tudies have been car r ied out with inhibitor Bill from peanut, using 1H NMR (Koyama et al., 1986), and with the complex of bovine trypsin and inhibitor ABI from azuki beans (Tsunogae et al. , 1986). The first study indicated that cleavage of the two r e a c t i v e s i tes resul ted in conformational changes tha t exclusively af fec ted the disulphide loops containing the react ive sites. From the second work it was concluded that, similarly to other families of inhibitors, both the primary and the secondary eontact regions ("front side" and "back side" of the ring) were essential for the inhibitory interaction.
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Figure. 3. The primary sequenee of PI1 from peanut, aligned to highlight the three-dimensional domain structure and infernal homology, aceording to Suzuki et al. (1987). The reactive sites are marked by ásterisks and thick Unes show disulphide bridges.
Genetics and Molecular Cloning
There is a surprising lack of gene t ic studies concerning the intraspecific variation and genome organization of the multi-gene families encoding the BBI family of inhibitors. As already pointed out , such s tudies would g r ea t l y help our understanding of the evolution of this protein family. The molecular cloning and analysis of a gene coding for BBI has been reported by Larkins and co-workers (Hammond et al., 1984). They constructed and characterized a cDNA clone cor responding to BBI and, using this clone as a probé , determined that the BBI gene was present in only one or two copies per genome. They did not find cDNA clones for other members of the family. Sequenee analysis of a genomic clone revealed that it was similar, but not identical, to the cDNA sequenee and that it had no introns. Additional cDNA and genomic sequences will have to be analysed to explain the s igni f icance of the observed variat ion between the two clones. It is of interest to note that while the BBI cDNA probé did not hybridize with other genes of the BBI family in soybean, it was able to do so with homologous genes from mimosa and redbud. Based on the DNA sequenee, it seems tha t the ini t ia l translation product must contain a short leader sequenee. An estímate of divergence time of 310 million years was made for the first and second domains of BBI based on the divergence of their nucleotide sequences.
Physiology
The BBI and its homologues do not appear to accumulate in any plant tissue other than the developing seed (Hwang et al., 1978). BBI mRNA accumulates early during the mid-maturation stage and reaches a steady state later in development (Foard et al., 1982; Hammond et al., 1984). The quantity of protein accumulated is of the same order as that for the Kunitz inhibitor.
During germinat ion, at leas t par t of the inhibitor is rapidly released from the seed within the first 8h of imbibition (Hwang et al., 1978; Wilson, 1980; Tan-Wilson and Wilson, 1982; Horisberger and Tacchin i , 1982). The rapid r e l éase suggests i ts elution from a b a r r i e r - f r e e pool, which is in l ine with the fac t t ha t BBI is d e t e c t e d in the in terce l lu lar space prior to germination but is absent from this space in 4-day oíd seedlings. Besides this reléase, it has now been documented in azuki bean (Yoshikawa et al., 1979b), soybean (Madden et al., 1985), and mung bean (Lorensen et al., 1981; Wilson and Chen, 1983) tha t this type of inhibitor undergoes a specific and extensive proteolytic processing during the early stages of ge rmina t ion and seedl ing g rowth . The significance- of these phenomena is unclear, but they seem to exelude a reserve role for this protein family.
CEREAL TRYPSIN/a-AMYLASE INHIBITOR FAMILY
This protein family includes a wide range of components whose structural relationships, often unsuspected, have only recently been fully demonstrated. Inhibitors of heterologous a-amylases were first described in wheat endosperm by Kneen and Sandstedt (1943). Years later, work on the main wheat albumins (Fish and Abbot, 1969; Ewart, 1969; Feillet and Nimmo, 1970; Sodini et al., 1970; Cantagalli et al., 1971, and others), whieh were found to be identical to some of the inhibitors, led to the realization that the inhibitors represent a major part of the álbum in and globulin f rae t ion of the endosperm. The first trypsin inhibitor of this family was isolated from barley endosperm by Mikola and Suolinna (1969), but its relationship to the a-amylase inhibitors was discovered only recently (Odani et al . , 1982, 1983a). The CM- proteins from wheat, barley, and rye, which were so designated because of their solubility in chloroform:methanol mixtures (Garcia-Olmedo and Gareia-Faure, 1969; García-Olmedo and Carbonero, 1970; Rodriguez-Loperena et al., 1975; Salcedo et al., 1978b, 1982; Paz-Ares et al. , 1983a), were eventually found to be members of the t rypsin/a-amylase inhibitor family, an observation that led to the discovery of new a-amylase and trypsin inhibitors (Shewry et al., 1984; Barber et al., 1986a,b; Sanchez-Monge et al., 1986b). Finally, homologous relationships of the cereal inhibitors were established with the 2S storage proteins from castor bean {Rieinus communis) (Odani et al., 1983c) and the Kazal secretory trypsin inhibitor from bovine páncreas (Odani et al., 1983b), thus showing tha t this prote in super-family is distributed beyond the plant kingdom.
Distribution and Inhibitory Properties
Inhibitors of heterologous a-amylases have been described in a number of species, although the available information for some of them is not as complete as that for those of wheat and barley.
F r a c t i o n a t i o n of a p a r t i a l l y pur i f i ed NaCl e x t r a c t by ge l f i l t r a t ion al lowed a c lass i f i ca t ion of the wheat inhibitors into three classes - monomeric, dimeric and tetrameric - which showed d i f fe ren t s p e c i f i c i t i e s : the d imer ic inhibi tor seemed to inhibit mainly a-amylase from human saliva, while all three inhibitors were active against a-amylase from the insect Tenebrio molitor (Petrucci et al., 1974; for a review see Buonocore et al., 1977). Purification and c h a r a c t e r i z a t i o n of ind iv idua l inh ib i to r s from wheat was undertaken at severa! laborat oríes (Shainkin and Birk, 1970; Saunders and Lang, 1973; Silano et al . , 1973; Petrucci et al., 1976, 1978; O'Donnell and McGeeney, 1976; O'Connor and McGeeney, 1981a; Maeda et al., 1982) and two types were characterized, respectively designated 0.28 and 0.19. The first type represented monomeric variants of about Mr 12,000 that were more act ive against insect a-amylases than against the human salivary or pancreatic ones. Those of the second
type were dimeric of about Mr 24,000 and were more effeetive against insect enzymes. The dimers eould be dissociated into Mr 12,000 subunits that were initially thought to be different (Silano et al., 1973), although more recent evidence suggests that they might be mixtures of homodimers (Maeda et al., 1985). Both types seem to have one moleeule of carbohydrate per subunit, which might be important for their inhibitory act ivi ty (Silano et al., 1977; Petrucci et al., 1978), and to share a number of physicochemical eharacter is t ics (Silano et al . , 1973; Silano and Zahnley, 1978). Their interaction with the enzyme was also studied and stoichiometries of 2:1 for 0.28 and 1:1 for 0.19 were proposed. The sugar moieties were suspected to be essential for the formation of the complex, which would explain the reversión of the inhibition by maltose (Silano et al., 1977; and Buonocore et al., 1980).
The w h e a t t e t r a m e r i c i n h i b i t o r h a s b e e n l e s s a c t i v e l y investigated. O'Connor and McGeeney (1981a) isolated an inhibitor with an apparent Mr of 63,000, which dissociated into subunits of Mr 14,000 and 15,000 with no carbohydrate, while Buonocore et al. (1985) determined an apparent Mr of 48,000 by equilibrium sedimentation and separa ted four electrophoret ic bands, all of which seemed to be inhibi tory. According to these authors, this inhibitor was active against both insect and human a-amylases but not against those from b a c t e r i a {Bacillus subtilis) or from fungi (Aspergillus oryzae). A mino acid analyses and circular dicroism spectra of the subunits indicated tha t they were similar to those of the monomeric and dimeric inhibitors. Recent reconstitution experiments have shown that proteins CM2, CM3 and CM16 from tetraploid wheat and the same proteins plus CM1 and CM17 from hexaploid wheat are components of the t e t r amer ic inhibitors (Sanchez-Monge, unpublished), thus identifying the subunits of the inhibitors described by Buonocore et al. (1985).
An extensive survey of the inhibitory properties of the three size classes of inhibitors versus a-amylases from 18 insect, 23 marine, and 17 avian and mammalian species was carried out by Silano et al. (1975). It was found that the monomeric class was mostly effeetive a g a i n s t t he insec t enzymes , espec ia l ly aga ins t those of grain predators, the Mr 24,000 class was more effeetive against marine, avian and mammalian a-amylases, and no clear pattern was observed for the te trameric class. These findings probably reflect the properties of the predominant components within each class, and not the proper t ies of ' speci f ic purified components . Thus Orlando et a l . (1983) found inhibition of the B.subtilis a-amylase by components of the 0.19 famiíy, and the monomeric fraction from Aegilops speltoides also inhibits the human enzyme (Bedetti et al., 1974).
The existence of a-amylase inhibitors corresponding to the three size classes has been recen t ly demons t ra ted in barley, and the previously character ized proteins CMa, CMb and CMd have been identified as subunits of the tetrameric inhibitor (Sanchez-Monge et a l . , 1986b). These th ree pro te ins have been shown to be truly homologous to those of the monomeric and dimeric inhibitors from wheat and only one of them, CMa, has been found to be active by i t se l f (Barber e t a l . , 1986a, b; Sanchez-Monge et a l . , 1986b). Reconstitution experiments indicated that all binary mixtures were
a c t i v e , including those b e t w e e n i n a c t i v e subuni t s , and t h a t t he mix ture of t he t h r e e subuni t s had t h e h ighes t specif ic ac t iv i ty (Sanchez-Monge et al . , 1986b). The barley t e t r amer i c inhibitor was act ive against the oc-amylase from T.molitor but showed no effect against salivary a-amylase. Evidence has also been obtained for the presence of a te t ramer ic inhibitor with similar subunits in the wild barley Hordeum chilense (Sanchez-Monge et al-, 1987).
A barley protein designated CMb' has been recently characterized as the subunit of a homodimeric a-amylase inhibitor that seems to be active against a-amylase from T. molitor but not against the salivary one (Sanchez-Monge, unpublished).
The only reported inhibitor from this family which has been found to be bifunct ional was isola ted from ragi {Eleusine coracana) by Shivarat and Pa t t ab i r anam (1980, 1981). This inhibitor is able to inhibit a-amylase and trypsin independently and is able to form a ternary complex with the two enzymes. The a-amylases of porcine páncreas, human páncreas and human saliva were inhibited in the ratio 5:5:1. A probable isoform of th is inhibi tor was la ter isolated by Manjunath et al. (1983).
O l i g o m e r i c i n h i b i t o r s of a - a m y l a s e have been i d e n t i f i e d in Phaseolus vulgaris (Pick and Wober, 1978; La jólo and Finardi- Filho, 1985). There is no evidence tha t the Phaseolus inhibitor is related to the cereal ones, whereas the amino acid composition of that from black bean is quite untypical of this group.
Other a -amylase inhibi tors , whose assignment to this family is s t i l l u n c e r t a i n , a r e h e t e r o d i m e r s wi th t h e subun i t s l inked by disulphide bridges; they have been found in rye (Granum, 1978), pearl millet (Chandrasekher and Pattabiranam, 1985), sorghum (Moideen Kutty and Pat tabi ranam, 1986) and Echinocloa frumentacea (Moideen Kutty and Pattabiranam, 1985). Finally, a 24,000 Mr heterodimer has been described in Setaria itálica that is made up of non-covalently linked subunits of Mr 12,000 and 16,000 (Nagaraj and Pattabiranam, 1985). More structural information about these inhibitors will be needed for their proper classification. It is not unlikely that at least some of them might have to be included in a new class.
Besides the t ryps in inhibi tor from rag i , which is bi funct ional , o the r t ryps in inh ib i to r s 'from th i s group have been ident if ied in barley, maize, rye, rice, and probably in wheat and sorghum.
The barley inhibitor was first identified by Mikola and Suolinna (1969), who isola ted a M r 14,100 pro te in from endosperm ac t ive against t rypsin and inac t ive agains t chymotrypsin, papain, subt i lo-p e p t i d a s e A, peps in , b a c t e r i a l or fungal p ro te inases , as well as against the endogenous proteinases from green malt. Antibodies raised against this protein did not react with inhibitors from barley embryo or with wheat endosperm extract; cross-reactivity with rye endosperm e x t r a c t was observed (Mikola and Kirsi, 1972). A similar inhibitor was purified by Boisen (1976) from a different genetic stock. The inhibitor was sequenced by Odani e t a l . (1983a), who did not find activity against elastase or a-amylases from various sources.
Inhibitors which are probably related to that from barley have been characterized in wheat endosperm (Shyamala and Lyman, 1964; Boisen and Djurtoft, 1981a; Mitsunaga et al., 1982). At least four different species seem to be present in this tissue, which inhibit trypsin at a 1:1 ra t io and chymotrypsin in a non-stoichiometric manner. They have similar heat stability and isoelectric points to tha t of barley, and thei r repor ted amino acid compositions are compatible with interspecific homology, although they are moderately divergent.
A trypsin inhibitor which seems to be closely related to that of barley has been characterized in rye (Polanowski, 1974; Boisen and Djurtoff, 1981b; Chang and Tsen, 1981a,b). This inhibitor is also weakly active against chymotrypsin.
The trypsin inhibitor from maize has been well characterized. It was first isolated from the opaque-2 mutant as a Mr 11,000 protein which inhibited trypsin by forming a 1:1 complex and was inactive against chymotrypsin (Swartz et al., 1977). The complete amino acid sequence of this inhibitor was obtained and found to be homologous to t h e ba r l ey one (Mahoney e t a l . , 1984). S imi lar or i d e n t i c a l inhibitors have been identified in different types of maize (Johnson et al., 1980) and in teosinte (Corfman and Reeck, 1982). An of the Hageman factor fragment for which three variants were isolated from maize by Hojima et al. (1980a) is probably identical to the trypsin inhibitor.
An inhibitor from rice bran of about Mr 14,500 which forms a 2:1 complex with trypsin (Tashiro and Maki, 1979; Maki et al., 1980) and one from sorghum (Filho, 1974) seem to be also of the barley type.
No inhibitory properties have been described for the 2S storage proteins from Ricinus (Sharief and Li, 1982), Brassiea (Crouch et al . , 1983), Lupinus (Lilley and Inglis, 1986) or Bertholletia (Ampe et al., 1986).
Structure and Evolution
Total or par t ía l amino acid sequences have been obtained for different members of this protein family either by direct protein sequencing or indirectly from cloned cDNAs (Fig. 4). Percentages of homology calculated for all possible binary comparisons are present ed in Table 5. A higher number of members have been characterized in wheat and barley than in any other species. Evolutionary implications of the observed homologies are best understood if they are considered toge ther with the "in vitro" ac t iv i t i e s of the proteins and the chromosomal locations of their corresponding genes in both species. The distribution among chromosomes of the multi-gene family encoding this group of proteins has been investigated through the analysis of wheat aneuploids and barley-wheat addition lines. In the case of wheat and barley, a number of loci can be postulated based on the general observation that there is greater homology between a given
Table 5. Binary comparisons (% homology) of members of the cereal a-amylase/trypsin Inhibltor famlly. Homology was calculated for common segments of the sequences that appear 1n Fig. 4. Abrevlations are as in Fig. 4.
PROTEINS
SPECIES [
WHEAT
WHEAT
BARLEY
WHEAT
BARLEY
WHEAT
WHEAT
BARLEY
BARLEY
BARLEY
WHEAT
WHEAT
WHEAT
BARLEY
BARLEY
BARLEY
BARLEY
RAGI
MAIZE
PEA
CA.BEAN
RAPE
BR.NUT
LUPIN
cow
CM16
CM17
CMb
CM3
CMd
CM2
CM1
CMa
CMc
C13
0.53
0.19
0.28
C44
C23
C38
CMe
RBI
MTI
PA
2SC
2SR
2SB
2SL KB
CM16
CM17
CMb
78 83
70
m x: CJ
45
37
52
CJ
40
36
47
70
CVI
x:
45
37
45
50
47
x: •
41
37
41
39
37
86
•o x: CJ
38
35
38
43
40
86
82
o z:
33
33
33
38
43
72
69
78
CJ
35
37
35
34
29
40
43
41
34
n
O
27
26
30
41
30
38
34
31
31
22
en
o
27
26
30
41
30
34
31
28
28
23
94
oo CVJ
o
27
30
30
30
23
37
33
30
30
23
58
56
«3-
32
30
29
26
22
39
35
32
32
22
45
44
63
ro CVJ CJ
22
25
26
32
35
32
32
32
35
30
26
26
25
24
00
CJ
23
27
28
24
22
32
O) x: CJ
38
37
45
46
43
54
53
50
45
36
28
30
25
23
42
34
CQ OH
43
37
47
45
39
52
48
48
43
34
29
29
30
26
49
35
56
SE
37
33
43
48
35
41
41
38
33
38
30
30
29
26
47
31
51
64
19
20
22
16
22
22
19
19
22
19
19
19
22
27
22
7
27
22
22
CJ
CVI
17
19
17
17
16
21
21
24
17
15
19
20
19
17
18
16
23
19
16
11
cu CVI
16
18
16
13
13
13
13
13
13
12
15
16
15
14
13
15
14
14
12
14
35
CQ
CVI
27
19
23
27
27
27
23
23
27
16
17
18
17
16
19
17
18
20
15
12
30
21
to
13
19
13
24
19
17
17
21
20
16
21
23
19
16
16
16
18
18
18
8
28
22
22
2
17
11
11
10
8
16
15
22
21
24
8
9
6
14
protein from one genome and the appropriate one from a different genome than between that protein and any other eneoded in the same genome. The proposed loci are Usted together with the in vitro ac t iv i t i e s of the corresponding prote ins in Table 6. These data indica te that this dispers'ed mult i-gene family has originated both by translocation and by intrachromosomal duplication, and that most if no t a l l of t h e d i s p e r s i ó n must have o c e u r r e d p r i o r t o t h e branching-out of the barley genome from the diploid geno mes included in allohexaploid wheat. The clearest case of an intrachromosomal duplication is that of the Cma and Cmc loci in chromosome 1 of barley, whose corresponding proteins are closer to each other than to any other eneoded in the same genome. Nevertheless, protein CMa shows an even higher s imi lar i ty to proteins CM1 and CM2, which are respectively eneoded in chromosomes 7D and 7B of wheat and, as CMa, are subunits of the tetrameric a-amylase inhibitors. In contrast, CMc is a trypsin inhibitor which shows a much greater divergence from CMe, the other trypsin inhibitor eneoded in chromosome 3, than from CMa (45% homology vs 78%).
Figure 4. (opposite) Alignment of a mino acid sequences of members of the cereal a-amylase/trypsin inhibitors family. Wheat CM-proteins CM1, CM2, CM3, CM16 and CM17 are subunits of tetrameric ot-amylase inhibitors (Shewry et al., 1984; Barber et al., 1986a); barley CM-proteins CMa, CMb and CMd are subunits of tetrameric ct-amylase inhibitors (Barber et al., 1986b) and CMc and CMe are trypsin inhibitors (Shewry et al., 1984; Barber et al., 1986a; Odani et al., 1983a; Lázaro et al., 1985); 0.19 and 0.53 are wheat dimeric ot-amylase inhibitors (Maeda et al., 1985) and 0.28 is a wheat monomeric a-amylase inhibitor (Kashlan and Richardson, 1981); C13, C23, C38 and C44 are a mino acid sequences deduced from barley cDNA clones pUP13, pUP23, pUP38 and pUP44, respectively, the latter eorresponds to a dimeric a-amylase inhibitor (Paz-Ares et al., 1986; Lázaro, unpublished); RBI is an a-amylase/trypsin inhibitor from ragi, Eleusine eoracana (Campos and Richardson, 1983); MTI is a trypsin inhibitor from maize (Mahoney et al., 1984); PA is a sulphur-rich pea álbum in (Higgins et al., 1986); 2SC, 2SR, 2SB and 2SL are 2S storage proteins from Rieinus communis (Sharief and Li, 1982), Brassica napus (Crouch et al., 1983), Ber thol le t ia excelsa (Ampe et al., 1986), and Lupinus angustigolius (Lilley and Inglis, 1986), respectively; KB is the bovine Kazal inhibitor (Greene and B artelt, 1969). Gaps introduced for the alignment are indicated (-). Vertical arrows (y) indícate the reactive bonds of the trypsin inhibitors. The three sequence blocks approximately correspond to the three domains defined by Kreis et al. (1985). The N-terminal positions of the second chain of the two chained 2S globulins are indica ted by a vertical Une (\). Conserved positions are boxed. Unidentified residues are indicated by an asterisk (*).
Over a dozen different sequences of this family nave been detected as abundant proteins and/or mRNAs in the endosperm of barley (Salcedo et al. , 1984; Paz-Ares et al . , 1986 and unpublished), which is a diploid species, whereas the number of variants per genorne in wheat s e e m s t o be l o w e r , p o s s i b l y as a r e s u l t of d i p l o i d i z a t i o n (Aragoncillo et al., 1975; Sanchez-Monge et al., 1986a).
Because of the complexity of this protein family, as has been ascertained in wheat and barley, the evolutionary implications of the limited sequence information available in other species must be drawn with caution. Thus, the bifunctional inhibitor from E. eoracana has the highest homology with the maize trypsin inhibitor, followed by b a r l e y t ryps in inh ib i to r CMe, t he ba r l ey p r o t e i n encoded by cDNA clone pUP-23, and some of the wheat and barley subunits of the tetrameric a-amylase inhibitors, but proteins with higher percentage of interspecific homology may yet be found in these species (Table 5). Protein PA from pea seeds is quite distant from the wheat and barley proteins, showing the highest homology with barley trypsin inhibitor CMe and with the barley protein encoded by clone pUP44. It should be pointed out tha t in this and subsequent cases of weak homology (20%-30% range), most of the homology is due to highly conserved or invariant positions, most notably the cysteines (Fig. 4).
53
- - Q N V ¥ [Q]Q M V E t? M R R R[Q]Q[E |G ! - G RIEIA
156
There is considerable divergence within the group of 2S storage proteins, which have no known inhibitory activity, and between this group and the r e s t of the sequenced prote ins . The in t ra-group homology is in the 20%-35% range , whereas homology with the nearest cereal components is in the 18%-27% range (Table 5).
Table 6. Chromosomal locations of genes and inhibitory act ivi t ies of the a-amylase/ trypsin inhibitors from wheat and barley.
Chromosome group
3
?
4
6
7
1 *
7
* Hexapl»
Genome*
Did
B
D
H
H
A
0
H
A
H
D
B
D
H
H
H
wheat
Sp
IT .
ecies
W
W
B
B
W
W
B
W
B
W
W
W
B
B
B
Prote in grouped by l o c i I n h i b i t o r y a c t i v i t y
I (0.53)
I I I (0.19)
CMb' (c44)
CMe
CM16
CM17
CMb
CM3
CMd
I I (0.28)
CM2
CM1
CMa
CMe
a-amylase (d imer ic )
t r y p s i n
a-amylase ( te t ramer i c )
a-amylase ( te t ramer i c )
a-amylase (monomeric)
a-amylase ( te t ramer i c )
t r y p s i n
C13,C23,C38 ?
aest ivum): qenomes AABBDD and bar ley (H. vul
References**
a ,b ,c
a,b,c
d
e , f
a,b
a,b
e
a,b
e
b,c ,g
a,b,h
a,b,h
e
e
i . j
qare) : genomes HH. Barley chromosome 1 homologous to chromosome group 7 of wheat.
** a) Aragoncillo et a l . (1975); b) Fra-Mon et a l . (1984); c) Sanchez-Monge et a l . (1986a); d) Sánchez-Monge and Lázaro (unpublished); e) Salcedo et a l . (1984); f) Hejgaard et a l . (1984); g) Pace et a l . (1978); h) García-Olmedo and Carbonero (1970); i) Paz-Ares et a l . (1986); j ) Lázaro (unpublished).
The Kazal secre tory trypsin inhibitor from bovine páncreas shows s ign i f i can t homology only wi th t h e t ryps in inhibi tors from maize , ragi and barley CMe (Table 5). The three domains proposed for barley t r y p s i n i n h i b i t o r CMe by K r e i s e t a l . (1985) a r e a p p r o x i m a t e l y i n d i c a t e d in F i g . 4 . The r e a c t i v e s i t e is jus t a t t h e r i g h t - h a n d border of t h e f i rs t domain and the sequence -Pro-Arg-Leú- , which cor responds to t h e - P 2 - P l - P ' l - res idues , has been conserved in the t h r e e pro te ins with ant i- t rypsin ac t iv i ty which have been completely sequenced (Fig. 4). Otherwise, the región around the trypsin reactive s i t e a p p e a r s a s e x t r e m e l y v a r i a b l e t h r o u g h o u t t h i s fami ly , wi th numerous deletions and/or insertions.
298
A weak but significant homology has been found between the alluded domains of these proteins and similar domains present in prolamins such as a-gliadin, Bl-hordein, ~<j~ -secalin, HMW-prolamin, and other cereal prolamins, in which these domains are separated by stretches of repeated sequences (Kreis et al., 1985). The sequence information accumulated since that finding further supports the proposed homology as better fitting comparisons can be made.
The sequences in Fig. 4 were searched for internal repeats because the homology of the Kazal inhibitors with the C- terminal part of these pro te ins , and the double-headed nature of the bifunctional inhibitor from ragi and of the trypsin inhibitor from rice bran, suggested a possible duplication. Only in the case of the monomeric ot-amylase inhibitor (0.28) a weak homology was found between the N-terminal and the C- terminal domains:
18 L T G C R A M V K L Q C V G S Q V P E
... . . . . . . . 1 1 / 2o L P G C R K E V M K L T A A S V P E
96
Genetics
Apart from the studies already discussed regarding the chromosomal locations of genes encoding proteins of this group, research on other aspects of their genetic control and regulation has also been carried out . The considerable sequence divergence found for this protein family within a geno me is in sharp contrast with the low variability which has been observed for the different members of the group. In a survey of tetraploid and hexaploid wheat cultivars, no variants were found for proteins CM1 and CM2, and only a rare allelic variant of protein CM3 was present in a small group of closely related cultivars (Garcia-Olmedo and Garcia-Faure, 1969; Garcia-Olmedo and Carbonero, 1970; Rodr iguez-Loperena et a l . , 1975; Salcedo et a l . , 1978a). Similarly, in a wide-ranging survey of Hordeum vulgare cultivars and H. spontaneum accessions, proteins CMa and CMc were invariant in the cul t iva ted species and presented ra re var ian ts in the wild one, protein CMd was invariant in both species, and proteins CMb and CMe had a l l e l i c v a r i a n t s fn bo th s p e c i e s (Sa l cedo e t a l . , 1984; Molina-Cano et al., 1987). In the case of the trypsin inhibitor CMe, two variants, designated CMe2/CMe2', appeared in over 40% of the samples and were jointly inherited and codominantly expressed with respect to CMe (Salcedo et al., 1984).
Other surveys have been concerned with the variation of the inhibitory act ivi ty against trypsin (Kirsi, 1973; Kirsi and Ahokas, 1983) and against a-amylases (Bedetti et al., 1974). The considerable v a r i a t i o n in the l evé i s of a c t i v i t y ind ica ted t h a t these were probably due not only to changes of the specific activity among variants but also to extreme variations in the expression levéis of the corresponding genes.
The regulation of the expression of genes from this family has been studied in some detail in a number of cases. Salcedo et al. (1978a) demonstrated that the net accumulation of protein CM3, now known to be a subunit of the tetrameric a-amylase inhibitor in wheat, and tha t of i ts al lel ic variant CM3', varied l inearly with gene dosage but the amount of CM31 for a given dosage was about half of that of CM3. The expression level eosegregated with the structural var ia t ion indicat ing tha t it was dependent on cis-acting genetic e l e m e n t s . A gene-dosage compensation effect was described by Aragoncillo et al. (1978) for proteins CM16 and CM17, which are also equivalent subunits of the tetrameric a-amylase inhibitor in wheat. Although the net output of each of the proteins varied linearly with gene dosage , the amount of pro te in a t a given dosage of i t s structural gene was significantly higher if the chromosome carrying the gene for the other protein was absent. This suggests some degree of coordinated expression mediated by trans-acting genetic elements of the genes encoding subunits of the tetra mers.
The effeets of high-lysine mutations on the expression levéis of different genes from this family have been investigated (Salcedo et al., 1984; Lázaro et al., 1985). The most notable effect concerns the gene for the trypsin inhibitor CMe, whose product is not detected in mutant Riso 1508 and is at a much lower level in Hiproly, as has been conf i rmed r e c e n t l y (Sa lcedo , unpubl ished) . In both cases , the mutations affect loci in chromosome 7, while the structural gene for the inhibitor is in chromosome 3 (see Lázaro et al., 1985). Just the opposite ef fect has been described for the high-lysine mutation opaque-2 of maize, which greatly increases the corresponding trypsin inhibitor (Halim et al., 1973).
Physiology
Tissue localization of the barley trypsin inhibitor(s) was found to be restricted to the starchy endosperm and the aleurone layer, as they were not detected in husks, coleoptiles, leaves or roots (Kirsi and Mikola, 1971). Similarly, Buonocore et al. (1977) concluded that the a-amylase inhibitors were endosperm-specific. Synthesis of this protein family seems to precede that of the bulk of reserve proteins and starch. Thús, Kirsi (1973) detected the barley trypsin inhibitor at about 5 days after anthesis (daa) and found that most of it was accumulated between 10 and 23 daa. He did not find a response to nitrogen fertilization. The a-amylase inhibitors were detected at 8 daa (Pace et al., 1978). In a study of in vivo and in vitro synthesis of these proteins in barley, Paz-Ares et al. (1983b) concluded that synthesis took place between 10 and 30 daa, with a peak between 15 and 20 daa. These proteins were synthesized by membrane-rbound polysomes as precursors of higher apparent Mr (13,000-21,000) than the mature proteins (12,000-16,000). The largest in vitro product (21,000) was found to be the precursor of subunit CMd. Accordingly, a putative leader sequence has been found in those cDNA clones of this family that were large enough to include the 5'-end of the mRNA sequence (Paz-Ares et al., 1986).
The subcellular location of the different members of this family has not been inves t iga ted . Indirect evidence of association with stareh granules has been reported (see Buonocore et al., 1977) and indeed at least some of the inhibitors show affinity for insoluble stareh (O'Connor and McGeeney, 1981b). However, Paz-Ares et al. (1983b) f o l l o w i n g d i f f e r e n t ho m o g e n i z a t i o n and s u b c e l l u l a r f ract ionat ion procedures consistently found these proteins in the supernatant , indicating that their association with the particulate f r ac t ion was l ab i l e if it ex i s t ed a t a l l . On the o ther hand, Zawistowska and Bushuk (1986) recovered CM-proteins in gluten preparations, where they seemed to have a considerable amount of bound polar lipids.
During germinat ion, the anti-trypsin activity disappears ra ther abruptly af ter remaining s table for 2-3 days (Kirsi and Mikola, 1971). Similarly, rapid disappearance with the onset of germination have been observed for the ot-amylase inhibitor (Pace et al., 1978). These observa t ions suggest tha t these inhibitors do not play a specific role during germination.
POTATO INHIBITORS I AND H FAMILIES
The families of the non-nomologous potato inhibitors I and II will be considered together because the two inhibitors play a joint role in the systemic response to wounding (see Ryan, 1984). They were first discovered in potato tubers, where they aecumulate throughout development and rep resen t a substant ia l fraction of the soluble protein, and later they were found to aecumulate in wounded tomato and potato leaves (Green and Ryan, 1972; Plunkett et al., 1982; Sánchez-Serrano et al., 1986; Cleveland et al., 1987).
Dístribution and Inhibitory Properties
Inhibitor I is an oligomer of Mr 41,000, made of protomers of M*. 8,100, with one disulphide bond and one reactive site each, which inhibits chymotrypsin- l ike enzymes (Ryan, 1984). Besides potato, tomato and other Solanaceae (Gurusiddaiah et al., 1972; Lee et al., 1986), homologues of this inhibitor have been found in plant species outside this family, such as broad bean (Svendsen et al., 1984) and barley (Svendsen et al. , 1980, 1982; Jonassen and Svendsen, 1982), and in a lower animal, the leech (Seemuller et al., 1980).
The inhibitor from broad bean (Vicia faba) has no cysteine and is act ive against microbial serine proteases, including subtilisin, with which it forms a 1:1 complex, and is inactive against chymotrypsin and trypsin (Svendsen et al . , 1984). The subtilisin inhibitors from Vigna unguiculata and Phaseolus vulgaris seem to be closely related
t o t h a t f rom Vicia faba a s j u d g e d f rom t h e a m i n o a c i d compositions (Vartak et al., 1980; Seidl et al., 1978, 1982). It is v e r y p r o b a b l e t h a t t h e c o n s t i t u t i v e t r y p s i n i n h i b i t o r s from buckwheat, reported by Kiyohara and Iwasaki (1985), are also members of this protein family.
Two inhibitors, CI-1 and CI-2, have been well charaeterized in barley. Both of them inhibit chymotrypsin and subtilisin, but CI-1 has a single reactive site for the two enzymes and CI- 2 has one reactive site for chymotrypsin and two for subtilisin (Jonassen and Svendsen, 1982). Neither of the inhibitors has cysteines.
Eglin, the inhibitor from the leech, has no cysteines and inhibits elastase and cathepsin G (Seemuller et al., 1980). A more distantly related inhibitor of this family has been identified in yeast (Maier et al., 1979).
Inhibitor II is a dimer of Mr 23,000, made of two protomers of Mr 12,000, with five disulphide bonds and two r e a c t i v e s i tes per monomer, which respectively inhibit chymotrypsin and trypsin (Ryan, 1984). The inhibitors from this family in tomato and potato are re la ted to two smaller trypsin and chymotrypsin inhibitors from potato tubers, PCI-I and PTI-I (Hass et al., 1982) and with another p r o t e i n a s e i n h i b i t o r f rom egg p l a n t ( R i c h a r d s o n , 1 9 7 9 ) . Wound-inducible an t i t ryps in ac t i v i t y has also been de tec ted in spec ies such as alfalfa, tobáceo, s t rawberry , cucumber, squash, clover, broadbean and grape (Walker-Simmons and Ryan, 1977). It is yet unknown if these inhibitors are homologues of potato inhibitors I or II and in some cases , such as that of the alfalfa inhibitors (Brown and Ryan, 1984), it is suspected that they might belong to entirely different groups.
Structure and Evolution
Available sequence information of the potato inhibitor I family is s u m m a r i z e d in Fig. 5. In p o t a t o e s , a mixture of 10 or more isoinhibitors are present and variation is observed at 16 out of the 84 positions of Jhe mature protein (Richardson, 1974; Richardson and Cossins, 1974; Graham et al., 1985a,b, Cleveland et al., 1987). The tomato sequence presented 80% homology with respect to the potato isoinhibitors (Fig. 5). As expected, lower homologies were found between the tomato inhibitor and those from barley (CI-1C, 25%; CI-2, 33%) and broad bean (35%). A remarkably low divergence (35% homology) was found for the animal inhibitor Eglin, whereas the homology of the yeast iñtiibitor (a16%) was rather weak (Fig. 5). The react ive sites seem to be located at exactly homologous positions (Richardson et al., 1977), except for CI-1C from barley, which has a second sub t i l i s in s i t e (Met30-Ser31) besides the s tandard one (Jonassen and Svendsen, 1982). In other inhibitor families, the th ree -d imens iona l s t r u c t u r e is s t ab i l i zed by disulphide bridges, which seem to be essential for their activity and, accordingly, they are highly conserved throughout evolution. Members of potato
IL-l
EIzIQ
LIE
( Z » V , S , 5 ) K K P E G V N T
Y P E P T E
F fv v le - n pj V T V I T
I P V V G L R L A
Figure. 5. Alignment of amino acid sequences of members of the potato inhibitor I family. TI-I is from tomatoes (Graham et al., 1985a); PI-I is from pota toes (Melville and Ryan, 1972; Cleveland et al., 1987); CI-2 and CI-1C are from barley (Svendsen et al., 1982); VSI is from broadbean (Svendsen et al., 1984); LIE is the inhibitor Eglin from leech (Seemuller et al., 1980); Y IB is from yeast (Maier et al., 1979). Vertical arrows (r) indícate the reactive bonds. Conserved positions are boxed.
inhibitor I family either have one disulphide bond or none and it does not seem to be essential for their activity. Indeed, the crystal and mo lecu l a r s t r u c t u r e of bar ley inhib i tor CI-2, which lacks c y s t e i n e , and of i t s complex wi th sub t i l i s in Novo have been determined and few conformational changes have been found between the free and the complexed inhibitor (McPhalen et al., 1985 and McPhalen and James, 1987).
Although members of* the inhibitor II family have been well characterized (Plunkett et al., 1982), direct sequencing methods have not allowed the determination of complete amino acid sequences, which have now been deduced from the corresponding cDNA clones in tomato (Graham et al., 1985b) and in potato (Sánchez-Serrano et al., 1986). Homology between the deduced sequences of tomato and potato is high in the región between positions 29 and 154, where only 19 differences appear (Fig. 6). The preceding signal sequence is 6 amino acids shorter (positions 21 to 28) in the tomato. As shown in Fig. 6, a high degree of homology was observed between the putative inhibitor II from potato, the two smaller proteinase inhibitor peptides from potato tuber, PCI-I and PTI, and a similar inhibitor from eggplant (Richardson, 1979; Hass et al., 1982; Sánchez-Serrano et al., 1986). These smaller peptides could be derived from different isoforms of
inhibitor II by further processing, as suggested by the fact that PCI-I is completely homologous to the appropriate segment of one of the two sequences deduced from potato cDNA (Sánchez-Serrano et al., 1986). As pointed out by Graham et al. (1985b), the full- length inhibitors have a duplicated-domain structure (Fig. 6). Based on the known position of the single reactive site of the eggplant inhibitor (Richardson , 1979), the p u t a t i v e r e a c t i v e s i t es of the larger inhibitors have been postulated at the homologous positions of the two domains (Fig. 6), which would explain the known specificities of the different inhibitors (Graham et al., 1985b).
SIGNAL SEQUENCE
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P I C T N C C A G Y K G C N Y Y S A N G A F I C E G
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Lil
133
IElzII
EElzlI
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Figure. 6. Alignment of amino acid sequences of members of the potato inhibitor II fámily. TPI-II is from tomatoes (Graham et al., 1985b); PPI-II is from potatoes (Sánchez-Serrano et al., 1986); PCI-I and PTI are from potatoes (Hass et al., 1982); EPI is from eggplant (Richardson, 1979). Vertical arrows (w) indícate reactive bonds. Horizontal arrows ( • ) indícate internal duplications. Conserved positions are boxed.
Genetics and Molecular Cloning
The chromosomal location of genes encoding the chymotrypsin inhibitors in chromosome 5 of barley and the 20-fold increase in their expression as a result of the high-lysine mutation in the loeus lys of chromosome 7 have been reported (Jonassen, 1980; Boisen et al., 1981; Hejgaard et al., 1984a). As already discussed, the same high-lysine mutation exerts the opposite effect on the gene encoding barley trypsin inhibitor CMe (Lázaro et al., 1985). The gene for an inhibitor from rye which cross-reacts with antibodies raised against the CI inhibitors from barley has been located in chromosome IR (Hejgaard et al., 1984b).
Although the variability of the concentration of the inhibitors in potato tubers has been investigated (Ryan et al., 1977), no other formal genetic studies seem to have been carried out concerning these two inhibitor famil ies . In con t ras t , considerable progress in the molecular cloning of these genes has been achieved in different species: cDNA clones for type-I inhibitors from tomato (Graham et a l . , 1985a) and barley (Williamson et al . , 1987) and for type-II i n h i b i t o r s f rom t o m a t o ( G r a h a m e t a l . , 1985b) and p o t a t o (Sánchez-Serrano et a l . , 1986) are now avai lable, together with genomic clones for type-I from tomato (Lee et al., 1986) and potato (Cleveland et al . , 1987) and for inhibitor-II from potato (Keil et al., 1986).
Comparison of the amino acid sequences, deduced from nucleotide sequences, with the N-terminal sequences of the native inhibitor-I proteins from tomato and potato indicates that in both cases a p recursor is coded which must yield the mature protein a f te r processing an N-terminal pre- and pro- sequences. The putative pre-or transit sequences are 23 residues long in both cases, whereas the pro-sequences would have 19 residues, 9 of which are charged in tomato and 16 residues, 6 of which are charged, in potato (Graham et al . , 1985a,b, Cleveland et al., 1987). The nucleotide sequences of several cDNAs corresponding to inhibitor CI-2 from barley have an open reading frame that corresponds exactly to the mature protein sequence. Although there is another ATG codon further 69 nucleotides upstream, the sequence between the two ATG codons would code for a typical signal peptide. An in-frame TAA stop codon is consistently p resen t and in vi t ro t rans la t ion s t a r t s in the downstream ATG (Will iamson et a l . , 1987). The open reading frames in cDNAs c o r r e s p o n d i n g to i n h i b i t o r - I I in t o m a t o and p o t a t o code for precursors of 148 and 154 amino acids, respectively. The N-terminal sequences of these precursors have all the features of typical leader sequences. The tomato leader sequence is six amino acids shorter, which accounts for the difference' in length of the precursors (Graham et al., 1985b; Sánchez-Serrano et al., 1986).
Inhibitor-I genes have two introns both in tomato and in potato, whereas a potato gene for inhibitor II has only one intron (Lee et al., 1986; Cleveland et al., 1987; Keil et al., 1986). The cDNA and genomic clones of the inhibitors have been searched for common features tha t could be re la ted to the regulation of the systemic
305
wound response. The two tomato proteinase inhibitor mRNAs share a v a r i a n t p o l y a d e n y l a t i o n s i g n a l ( A A T A A G ) a n d a c o n s e r v e d 3'-untranslated 10-base palindromie región, but these are not present in the potato mRNAs (Graham et al. , 1985a,b; Cleveland et al., 1987; Sánchez- Serrano e t a l . , 1986). An imperfect d i rec t repeat 100 bp long was found in the 5'-flanking región of the inhibitor I gene from tomato tha t was shared by other Lycopersicon species with inducible inhibitors, but not by the po ta to genes for inhibitors I and II (Lee et al . , 1986; Cleveland et al., 1987; Keil et al. , 1986). Homology of the 3 ' -untranslated regions of the proteinase inhibitor II genes from p o t a t o a n d an e x t e n s i n g e n e h a v e b e e n a l s o p o i n t e d ou t by Sánchez-Serrano et a l . (1987), but again this homology is not shared by all the inducible genes.
Physiology
Certa in species such as tomatoes , respond to wounding by insects or other severe mechanical damage within a few hours by accumulating p r o t e i n a s e i n h i b i t o r s in l e a v e s t h r o u g h o u t t h e p l a n t , i nc lud ing non-wounded parts (Green and Ryan, 1972; Plunkett et al., 1982;
QñllzL QüIlzLLL
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36
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ISIL 37 _ 50 P C K ' L N D D S L C G R K G
Figure. 7, Alignment of a mino acid sequences of the squash family of serine proteinase inhibitors. CMTI-I, CMTI-III and CMTI-IV are from Cucúrbita máxima; CPGTI-I, CPTI-II and CPTI-III are from C. pepo; CSTI-IIb and CSTI-IV are from Cucumis sativus (Wieczorek et al., 1985). Inhibitors CM-1 and CM-3 are from M o mordica repens (Joubert, 1984b) and TKTI is from Trichosanthes kirilowii (Tan et al., 1984). Conserved positions are boxed. Vertical arrows (y) indícate the reactive bonds.
Graham et a l . , 1986). The induction of the inhibitor proteins is mediated by a putative wound signal, called the proteinase inhibitor indueing f a c t o r (PUF), whose a c t i v i t y has been shown to be associated with fragments of the plant cell wall, apparently released at the wound site by endogenous endopolygalaeturonases (Bishop et al., 1984; Ryan, 1984). Following a single wound on a lower leaf of a tomato plant, mRNAs for the two inhibitors began to accumulate in leaves 2 to 4 hours later, and the proteins were detected with a further 2 hours lag. The levéis of the mRNAs reached a máximum at about 8 hours a f t e r wounding and then decayed with apparent half-l ifes of 10 hours; consecutive wounds had an accumulative effect. Synthesis of the inhibitors was found to be regulated at the level of transcription (Graham et al., 1986).
The wound-induced response has been investigated in transgenic tobáceo plants in which either the complete inhibitor-II gene from potato (Sánchez-Serrano et al., 1987) or a fusión of this gene with that of chloramphenicol acetyl transferase (Thornburg et al., 1987) had been introduced. The fact that these genes were wound-induced in the transgenic plants indicated that tobáceo has the capacity to regúla te the expression of these inducible genes. The sequences necessary and sufficient for wound-inducibility were located within lkbp upstream of the genes.
SQUASH TRYPSIN/HAGEMAN FACTOR INHIBITOR FAMILY
A new family of very small inhibitor (29-32 residues, 3 disulphide bridges) that are active against trypsin and Hageman factor have been described in the seeds of the Cucurbitaceae (Hojima et al., 1980b; Polanowski et al., 1980; Joubert, 1984b). Members of this family have been isolated and sequenced from squash (Cucúrbita máxima) by Wilusz et al. (1983), from zucchini (C. pepo var. Giromontia), summer squash (C. pepo), and cucumber (Cueumis sativus) by Wieczorek et al. (1985), and from Momordica repens by J o u b e r t (1984b). Fami l i e s of isoinhibitors have been found in all of these species, which seem to resul t both from múltiple s t ruc tu ra l genes and from proteolytic processing: while some Of the sequences are clearly different from each other, in other cases the heterogeneity is only due to the presence or absence of N-terminal peptides (Fig. 7). A double-headed inhibi tor from a Chínese medical herb , Trichosanthes kirilowii (Cucurbitaceae), which has 41 amino acid residues (Tan et al., 1984), is over 30% homologous to the shorter squash inhibitors and 26% homologous to soybean Bowman-Birk. family.
Disulphide bond posi t ions have been proposed, based on the s imi la r i ty of the squash inhibi tors with whea t -germ agglutinin (Siemion et a l . , 1984). The r e a c t i v e s i te is e i the r Arg-Ile or Lys-Ile in agreement with their trypsin specificity (Joubert, 1984b; Wieczorek et al., 1985). In spite of their small size, they are quite strong inhibitors. Up to now these inhibitors have been found only
within the Cucurbi taceae and it will be of in teres t to investígate their range of distribution and variation.
HOMOLOGY OF PROTEIN Z WITH al-ANTITRYPSIN AND RELATED PR OTEINS
Protein Z is a major barley endosperm albumin of Mr 43,000 which is present in both free and bound forms in the mature grain, where it seems to act as a storage protein and contains a substantial part of the grain lysine (Hejgaard, 1976, 1982; Hejgaard and Boisen, 1980; Giese and Hejgaard, 1984). Nucleotide and amino acid sequencing of cDNA clones have established the 180 C-terminal residues of this p r o t e i n ( F i g . 8) . This s e q u e n c e has been found to be 26-32%
a l - a n t i t r y p s i n , human otl-antichymotrypsin, mouse con t raps in and chicken ovalbumin The sequence homology and the specif ic bond corresponding to the reactive sites of possible inhibitory act iv i ty for protein Z,
a g a i n s t m i c r o b i a l or p a n c r e a t i c s e r i n e
homologous with human human a n t i t h r o m b i n III, (Hejgaard e t a l . , 1985). cleavage by trypsin at a the inhibi tors suggest a a l t h o u g h no a c t i v i t y proteinases has been found (Hejgaard et al., 1985).
PRQJzZ W al -AT Y
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Figure. 8. Comparison of a partial sequence from barley protein Z with al-antitrypsin (Hejgaard et al., 1985). Conserved positions are boxed.
RAGI 1-2 / MAIZE BIFUNCTIONAL INHIBITOR FAMILY
Bes ides t h e a l r e a d y d e s c r i b e d b i func t iona l inhib i tor from ragi (Eleusine coracana), a second un re l a t ed inhibi tor which was ac t ive a g a i n s t p o r c i n e p a n c r e a t i c a - a m y l a s e , was c h a r a c t e r i z e d in t h a t spec ies (Shivaraj and P a t t a b i r a m a n , 1980; Campos and Richardson, 1984). A Mr 10,000 protein was also purified from barley and found to be homologous to the ragi inhibitor 1-2 (Svensson et al., 1986; Mundy and R o g e r s , 1986) . Th i s p r o t e i n was c o n s i d e r e d as a p r o b a b l e a m y l a s e / p r o t e a s e inhibitor (PAPI) both because of this re la t ionship wi th t h e r a g i i n h i b i t o r and b e c a u s e of i t s weak but s ign i f ican t homology with Bowman-Birk t ype p ro t e inase inhibi tors (Mundy and R o g e r s , 1986), a l t h o u g h no t a r g e t e n z y m e was ident i f ied for it (Svensson et al . , 1986). Expression of the gene encoding PAPI, which has been s t u d i e d us ing a cDNA p r o b é , t a k e s p lace p r imar i ly in aleurone tissue during late stages of grain development. PAPI mRNA is p r e s e n t a t high levé i s in a l e u r o n e t i s sue of d e s s i c a t e d gra in , as well as in incubated aleurone layers prepared from rehydrated grain, PAPI protein being secre ted into the incubation médium (Mundy and Rogers, 1986).
The s e q u e n c e of a m a i z e p r o t e i n which is a p o t e n t in v i t ro inhibitor of bovine t rypsin and insec t a - amylase has been recent ly determined and found to be partially homologous with barley PAPI and r a g i 1-2 i n h i b i t o r ( R i c h a r d s o n e t a l . , 1 9 8 7 ) . The new m a i z e b i f u n c t i o n a l i n h i b i t o r showed e x t e n s i v e homology with the swee t p r o t e i n t h a u m a t i n II, p r e s e n t in t h e f r u i t s of Thaumatococcus danielli, and with a p a t h o g e n e s i s - r e l a t e d (PR) p ro t e in induced in t o b á c e o p l a n t s following infec t ion with tobáceo mosaic virus. This s t r ik ing observat ion suggest a possible inhibi tory function both for thaumat in II and the PR- protein (Richardson et al., 1987). Sequence al ignments showing the above described relationships are presented in Fig. 9.
INHIBITORS OF METALLOCARBOXYPEPTTDASES
Inhibi tors of meta l lo -ca rboxypept idases (CPI) were identified both in potato tubers (Rancour and Ryan, 1968) and tomato fruits (Hass and Ryan, 1980a), and were la ter found to accumulate in wounded leaves (Graham and Ryan, 1981). These inhibi tors a re polypept ides of Mr 4,200 which are unusually hea t s tab le . They are active against most a n i m a l c a r b o x y p e p t i d a s e s and i n a c t i v e aga in s t a l l p lant and most m i c r o b i a l c a r b o x y p e p t i d a s e s t e s t e d (Hass e t a l . , 1981). Inhibi t ion oceurs by a competit ive mechanism and results in the rapid removal of the ca rboxy- t e rmina l amino acid, whose c leavage does not lead to elimination of the inhibitory activity (Hass and Ryan, 1980b).
PAPL
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S C R T G D C G G N C [ | ] T G D C [ Ñ ] G
I C R T G D C G G
9 2 T |G Y G Rl A [ P N T L A ElYJ A L Q 1G Y 6 | " K | P P N T L A E F A L K R F l G R P P | T ] T L A E F f s l L
I L D G F N V P|Y S L V|D G F N (T]P MJE N i KIG F N v p MID
G C G C G C
IMi
5Si IPR THA
1 3 9 L I R - Q DfG~]vJ C N N A CJP jV F KJK D
G G C N NIPJC T V G G C N|D A C T V íls
E Y C C
E Y C C
V G S A A N N - T N G P [ G ] S
184
C P C P C P
1 8 5 D A Y S Y PIK D A Y S Y P|S D AÍF~lS Y ( v L
D D[A D D P D.K P
T P T
5 T F T C P S [ L ] F T C P - | T | V IT C P
N T I K l V V F C P N Y R V V F C P
|N Y R V I T I F C P
2 1 4
Figure. 9. a) Comparison of the complete sequences of PAPI, a probable a-amylase/proteinase inhibitor from barley (Mundy and Rogers, 1986), and RAI-2, the oc-amylase inhibitor 1-2 from ragi (Campos and Richardson, 1984), with homologous segments of MBI, a bifunetional a-amylase/trypsin inhibitor from maize (Richardson et al., 198 7). b) Alignment of the complete sequence of MBI with those of thaumatin (THA) and a pathogenesis-related protein induced by tobáceo mosaic virus (TPR), as reported by Richardson et al., 1987). Conserved positions are boxed.
Sequences of the highly homologous tomato and potato isoinhibitors have been aligned in Fig. 10. The structure of CPI-II from potato has been deduced by X-ray diffraction analysis at 2.5Á resolution of a complex of the inhibitor with carboxypeptidase A (Rees and Lipscomb, 1982). CPI-II, which has no helical structures or 3-pleated sheets, binds to the enzyme like an extended substrate. The Val38-Gly39 peptide bond is cleaved and Gly39 gets trapped in the binding pocket of the enzyme. In the case of the tomato inhibitor, which laeks an amino acid at position 39, inhibition also takes place, indicating that this amino acid is not required for activity.
A significant increase of CPI was observed in the upper, unwounded leaves 38h following wounding of the lower leaves from potato plants a n d t h i s r e s p o n s e i n c r e a s e d a s t h e p l a n t s g r e w o l d e r (Hollander-Czytko et al., 1985). The levéis of CPI accumulation in upper, unwounded leaves were increased by múltiple wounds in a similar manner to that described for proteinase inhibitors I and II of potatoes. Finally, the inhibitor was also found to accumulate in the vacuoles of unwounded, wound-induced leaves (Hollander-Czytko et al., 1985).
INHIBITORS OF THIOI/-PROTEINASES
Pro te in inhibitors of th io l -pro te inases , such as papain, ficin, bromelain, e tc . , have been identified in different plant species, but only a limited number of studies have been devoted to them and it is not yet possible to classify them into structural families.
A family of isoinhibitors from pineapple stems that competitively inhibit papain, ficin, and endogenous bromelain, is probably among the best charac te r ized of this group (Reddy et al. , 1975). They eons i s t of two chains with 41 and 10-11 amino acid res idues respectively (Fig. 11). It has been proposed that the two chains, which a r e l inked by d i su lph ide b r i d g e s , a r e g e n e r a t e d from single-chain precursors by excisión of a "bridge" peptide which links the amino-terminal of the A chain to the carboxy-terminal of the B chain (Reddy et al., 1975). *
PCPI E Q H A E H. .
TCPI E Q Y
D P
D P
I c
c
N
H
K P C
K P C
K
5
T
T
H
Q
D D C S G
D D C 5 G
A W
G T
F C Q A C W
F C Q A C W
A N S
R F
ÍR A
A
39
R T C G P Y V G
T C G P Y V
Figure. 10. Comparison of carboxypeptidase inhibitors from potatoes (PCPI) and tomatoes (TCPI) (see Hass and Ryan, 1981). PCPI has a varíant that differs at the N-terminal as indicated in the figure. Conserved positions are boxed.
An inhibitor of Mr 24,000 from the seeds of the Bauhinia tree is ac t ive against a wide range of prote inases : papain, ficin, bromelain, trypsin, chymotrypsin and pepsin (Goldstein e t al., 1973).
Two inhibitors from maize endosperm, with Mrs of about 13,000 and 9,500 r e s p e c t i v e l y , have been found to be a c t i v e agains t papain, ficin and bromelain (Abe e t al . , 1980), whereas an inhibitor isolated from dehulled rice seeds, which has an M r of about 12,000, was only a c t i v e a g a i n s t t h e f i r s t t w o e n z y m e s (Abe and Arai , 1985). An unstable papain inhibitor has been purified from the grain of foxtail millet (Tashiro and Maki, 1986).
INHIBITORS OF CARBOXYL PROTEINASES
A polyvalent , heat s table inhibitor of M r 27,000 which is ac t ive against ca theps in D, a carboxyl p ro t e ina se , has been isolated from p o t a t o e s ( K e i l o v a a n d T o m a s e k , 1 9 7 6 a , b ) . T h i s i n h i b i t o r simultaneously inhibits trypsin and chymotrypsin and has no effect on hog and ch icken peps in , rennin, or ca theps in E. The inhibition of cathepsin D and trypsin is reversible and competitive, with Ki valúes of 3.8 x 10M and 8.6 x 10M, respectively.
B r o a d s p e c i f i c i t y p r o t e i n a s e i n h i b i t o r s h a v e b e e n a l s o c h a r a c t e r i z e d in c u l t u r e d c e l l s of Scopolia japónica (So lanaceae ; Sakato et al., 1975). The inhibitors were shown to be polypeptides of M r 4 , 0 0 0 - 6 , 0 0 0 . T h e y a r e p o t e n t i n h i b i t o r s of p e p s i n , t r y p s i n , chymotrypsin , plasmin and kal l ikrein, and had no effect on papain. These inhibitors form stable complexes with trypsin and chymotrypsin in an equimolar ra t io . The inhibitory mechanism for both enzymes is non-competit ive.
1 41 ftri. D E Y K C Y C A D T Y 5 D C P G F C K K C K A E F G K Y I C L D L I S P N D C V K
1 11 B-2 T A C S E C V C P L Q
Figure.11. Sequences of the A and B chains of an inhibitor of thiol-proteinase from pineapple stem ( Reddy et al., 1975).
INHIBITORS AND PLANT METABOLISM
Most of t h e d e s c r i b e d i n h i b i t o r s a r e only a c t i v e aga ins t heterologous enzymes and it is hard to envisage their involvement in metabolic processes of their respective species, with the possible exception of a storage role which has been postulated for them in view of the i r spec ia l accumula t ion in r e se rve t issues and the dependence of the concen t ra t ion of some of them on ni trogen fertilization. Although such a role cannot be excluded, and might be even probable in some cases (especially in tissues like the cereal endosperm that are completely degraded during germination), in other cases a storage function is not likely because they are lost from the seed by diffusion (Tan-Wilson and Wilson, 1982) or undergo a specific p r o t e o l y t i c p r o c e s s i n g t h a t s e e m s to be d i f f e r e n t from the non-specific one of the true reserve proteins (Hartl et al., 1986; Yoshikawa et al., 1979b; Madden et al., 1985; Lorensen et al., 1981; Wilson and Chen, 1983).
Only those few inh ib i t o r s t ha t have been shown to a f fec t endogenous proteases and amylases are likely to play specific roles in plant metabolism.
P ro t e inase inh ib i to rs t ha t inhibit endogenous prote inases are found in barley (Mikola and Enari, 1970; Kirsi and Mikola, 1971), r ice (Horiguchi and Kitagishi, 1971), wheat (Preston and Kruger, 1976), maize (Reed and Penner, 1976; Abe et al., 1980), mung beans (Baumgartner and Chrispeels, 1976), Scots pine (Salmia and Mikola, 1980; Salmia, 1980), and alfalfa (Gonnelli et al., 1985). At least some of these inhibitors might be specific for thiol-proteinases (see Ryan, 1981). They are usually present in small amounts and rapidly disappear during early stages of germination. The disappearance of inhibitory act ivi ty is accompanied by a rise of the endopeptidase activity, but the kinetics of the two processes, both in mung bean (Baumgartner and Chrispeels, 1976) and in Scots pine (Salmia, 1980), suggest that they are not causally related. Furthermore, since the endogenous proteinase and the inhibitor have different subcellular locations, these authors have postulated that the inhibitors do not control the breakdown of storage proteins by the enzyme associated with the protein bodies, but probably play a protective function with respect to other cel lular components. As previously indicated, a possible role in the prevent ion of s tarch degradat ion has been proposed for the endogenous ot-amylase inhibitors (Mundy et al . , 1983), but as in the case of the endogenous proteinase inhibitors, no clear metabolic role has been fully- demonstrated for them.
INHIBITORS AND PLANT PROTECTION
Several lines of evidenee indícate that possibly a majority of the plant inhibitors that are active against heterologous proteinases and a-amylases play a protect ive role against the a t tacks of animal predators , insects, fungi, bacter ia , and viruses. The inhibition of hydrolytic enzymes from these organisms may be directly toxic or even lethal to them, or could affect their fitness in such a way that different feeding habits might evolve. The action of so me inhibitors on insect and microbial proteases has been repeatedly reported (Birk et al., 1963b; Applebaum et al., 1964a; Applebaum and Konijn, 1966; Peng and Black, 1976; Yoshikawa et al., 1976; Mosolov et al., 1976, 1979). Similarly, a-amylases of different origins have been tested against plant inhibitors (Applebaum et al. , 1964b; Silano et al. , 1975; Powers and Culbertson, 1982; Orlando et al. , 1983; Baker, 1987). Pa r t i cu l a r ly s ignif icant in the p resen t contex t was the observation that insects which were able to feed on a tissue like the cerea l endosperm, which has a high concentra t ion of a-amylase inhibitors, had significantly higher a-amylase activities than those that were not able to do so (Silano et al., 1975). More recently, G a t e h o u s e e t a l . (1986) have s t u d i e d the e f f e c t s on l a r v a e development of inhibitor preparations from wheat endosperm that were strong inhibitors of digestive a-amylases from larvae of Tribolium confusum, a storage pest of wheat producís, and Collosobruchus maculatus, a storage pest of legume seeds. While very high inhibitor concentrations were required to affect survival of larvae from the first species, quite low concentrations were effective against those of the second. The authors concluded that a detoxification mechanism mus t h a v e been f u n c t i o n i n g in t h e f i r s t c a s e , bes ides t h e considerably higher a-amylase ac t iv i ty , to account for the low sensitivity.
A more direct link of proteinase inhibitors with plant protection is represented by the already described systemic response which has been inves t iga t ed by Ryan and co-workers . Increased levéis of inhibitors are induced not only by the mechanical lesions caused by insects (see Ryan, 1984) but also following infection by fungi: Peng and Black (1976) demonstrated that while an incompatible race of Phytophthora. iníestans was able to elicit higher inhibitor levéis in tomato plants, a compatible one produced either a small transitory increase or no increase. The systemic response was two times greater with the incompatible race than with compatible ones.
The e f f e c t s of p r o t e i n a s e i nh ib i t o r s on i n s e c t s have been investigated in a number of cases. A possible involvement of these inhib i tors in the protect ion of barley against grasshoppers was postulated because leaf extracts of the less sensitive variety tested had a considerably higher ant i -chymotrypsin ac t iv i ty (Weiel and Hapner, 1976). Growth retardation and delayed pupation of corn borer larvae have been observed in feeding experiments with the Kunitz soybean trypsin inhibitor, while the maize trypsin inhibitor had no effect (Steffens et al., 1978). These and similar studies suggest but do not d e m ó n s t r a t e a p r o t e c t i v e role for the inhibitors. More
conclus ive e v i d e n c e has been ob ta ined for the r e s i s t a n c e of the cowpea (Vigna unguiculata) t o t h e bruchid bee t l e Callosobruchus maculatus. The only res i s t an t variety found in a screening of over five thousand var ie t i es had a higher level of prote inase inhibitors, whose a n t i m e t a b o l i c n a t u r e was d e m o n s t r a t e d in f eed ing t r i á i s (Gatehouse e t a l . , 1979). In compar i son , soybean trypsin inhibitor and l i m a b e e n t r y p s i n i n h i b i t o r w e r e r e l a t i v e l y i n e f f e c t i v e (Gatehouse and Boul ter , 1983). A convincing demonst ra t ion of the p r o t e c t i v e po ten t i a l of the cowpea inhibitor has been obtained by cloning the corresponding gene and expressing it in tobáceo under a const i tut ive p romote r . According to a recen t news item (Newmark, 1987), the transformed tobáceo plants have been tested against bud-and army worms, and in both cases, they fail to grow and eventually die. This approach will allow in the near future testing the possible protective properties of different inhibitors.
An excit ing new development concerns virus res is tance. It seems tha t r e s i s t ance to the cowpea mosaic virus in cu l t ivar Arlington d e p e n d s on t h e i n h i b i t i o n by a p l a n t i n h i b i t o r of t h e th io l -p ro te inase encoded by the vi rus , thus p reven t ing pro teo ly t ic processing of the viral polyprotein (see Ponz and Bruening, 1986). The recently reported homology of a PR-protein, induced by infection with tobáceo mosaic virus, with a bifunctional inhibitor from maize (Richardson e t a l . , 1987) is also of cons iderab le i n t e r e s t in the present context.
INHIBITORS AND NUTRITION
Due to their abundance in many plant tissues, which are used as food and feed componen t s , and to t he i r r e l a t i v e s t ab i l i t y versus hea t , enzymatic degradation, or ex t reme pH, the potential incidence of proteinase and a-amylase inhibitors on human health and on animal p r o d u c t i o n has b e e n e x t e n s i v e l y i n v e s t i g a t e d , l e a d i n g t o some controversies which are not quite sett led yet .
The effect of trypsin inhibitors has been recently reviewed by Liener (1986), who has pointed oüt the need for further research to remove the remaining unce r t a in ty concerning possible risks to humans. The páncreas of r a t s and chicks fed on raw soybeans is st imulated to produce more trypsin and beca mes enlarged as a result of hypertrophy and hyperp las ia . The sec re to ry ac t iv i ty is subject to inhibition by i n t r a l u m i n a l t ryps in th rough the inhibi t ion of the r e l éa se of the hormone c h o l e c y s t o k i n i n (CCK) - f rom the i n t e s t i n a l mucosa. The i n h i b i t i o n of t r y p s i n by t h e i n h i b i t o r r e s u l t s in an i n c r e a s e d reléase of CCK (Green and Lyman, 1972; Liddle et al., 1984). Most of t h e i n f o r m a t i o n r e l a t i n g t o t h e n u t r i t i o n a l e f f e e t s of t r y p s i n inhibitors has come from experiments with animáis and while the above described effeets appear also in mouse, hámster and young guinea pig, besides the rat and the chick, other animáis such as the dog, pig and calf do not d i sp lay t h i s s e n s i t i v i t y (see Liener , 1986). For th is
reason, extrapolation to man is not easy, although it has been shown tha t raw soybeans can inhibit completely trypsin and chymotrypsin in h u m a n p a n c r e a t i c j u i ce ( K r o g d a h l and Holm, 1981) . The K u n i t z i n h i b i t o r , which is h e a t l a b i l e , is i n a c t i v a t e d by incuba t ion with gas t r i c ju ice , while the Bowman- Birk inhibitor is both heat s table and insens i t ive to g a s t r i c ju ice , and t he re fo re , more likely to be present in processed soybean-containing foods and to survive passage through the s tomach. The infusión of Bowman-Birk inhibitor into the duodenum of human subjects has been shown to significantly increase the enzyme content of the pancreat ic juice (Goodale et al., 1985). A r epo r t ed outbreak of g a s t r o i n t e s t i n a l i l lness in individuáis who had consumed underproeessed soy p ro te in ex tender in a tuna fish salad would be a good i l lustrat ion of the potent ial risk for humans (Gunn e t a l . , 1980). In c o n t r a s t , a po t en t i a l l y benef ic ia l s ide-ef fec t has b e e n p o s t u l a t e d for t h e s e i n h i b i t o r s wi th t h e c la im t h a t d i e t s c o n t a i n i n g s o y b e a n s or s o y b e a n p r o t e a s e i n h i b i t o r s p r e v e n t t h e a p p e a r a n c e of t u m o r s in mouse skin t r e a t e d with carc inogens , of b r e a s t t u m o r s in r a t s s u b j e c t e d t o i o n i z i n g r a d i a t i o n s , and of spontaneous liver cáncer in C3H mice (see Troll et al., 1984).
Inhibitors of salivary and pancrea t i c ot-amylases in the diet could p o t e n t i a l l y a f f e c t s t a r c h d i g e s t i ó n . Indeed, Puls and Keup (1973) p r o p o s e d t h e u s e of t h e w h e a t a - a m y l a s e i n h i b i t o r a s a n an t ihyperg lycaemic agen t , based on t e s t s carr ied out in ra t s , dogs, and humans. A large number of commercial preparations of a-amylase inhib i tors or f ,starch b lockers" obta ined from kidney beans reached t h e m a r k e t . Even tua l ly , t he se p roduc í s were d iscredi ted and the i r d i s t r i b u t i o n d i s e o n t i n u e d on t h e bas is of t he i r i ne f f ec t i venes s in humans (Carlson e t a l . , 1983; Bo-Linn e t al . , 1982). A biochemical analysis of commercia l s ta rch blockers subsequently showed not only t h a t these p repa ra t ions were not par t icular ly enriched in a-amylase inhibi tors with r e spec t to the crude meal, but tha t they contained t r y p s i n i n h i b i t o r s a n d l e c t i n s , a s w e l l a s e n o u g h e n d o g e n ó u s , i nh ib i to r - in sens i t i ve a - amy la se to offset the i r a l leged e f fec t iveness as inhibi tors of s t a r c h diges t ión (Liener e t al . , 1984). More recen t s t u d i e s , u s ing b e t t e r c h a r a c t e r i z e d i n h i b i t o r s , s eem to conf i rm s i g n i f i c a n t e f f e e t s in r a t s and h u m a n s . An a - a m y l a s e i nh ib i t o r , prepared from black kidney beans (Phaseolus vulgaris), given to young r a t s by stomach tubing mixed with a s tarch meal was able to reduce in a dose -dependen t manner the hyperg lycaemia resul t ing from s t a rch u t i l i z a t i o n (Lajplo e t a l . , 1984). S imi la r ly , a pur i f ied whi te bean i n h i b i t o r w a s f o u n d t o be s t a b l e in h u m a n g a s t r o i n t e s t i n a l s ec re t ions , to slow d ie ta ry s ta rch digestión in vi t ro, and to rapidly i n a c t i v a t e a m y l a s e in t h e human i n t e s t i n a l lumen (Layer e t a l . , 1985) . The p o t e n t i a l u s e f u l n e s s of a - a m y l a s e i n h i b i t o r s in t h e t r e a t m e n t of d i a b e t e s and obes i ty c a n n o t be inferred from these studies but deserves reconsideration.
ACKNOWLEDGEMENTS
Ass is tance from D. Lamoneda , and Gran t s No. 2022/83 and No. 0193/85 from the Comisión Asesora de Inves t igac ión Cient í f ica y Técnica are gratefully acknowledged.
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