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Vol. 254, No. Ii, Issue of Septem ber 10, pp. 8447-8456, 1979Printed in U.S. A.

The Primary Structure of Calf Chymosin*(Received for publication, January 16, 1979, and in revised form, March 27, 1979)

Bent Foltmann, Vibeke Barkholt Pedersen, Dorothy Kauffman,+ and Grith WybrandtgFrom the Institute of Bioch emica l Genetics, University of Copenhagen, DK-1353 Copenhagen K, Denmark

The complete amino acid sequence of calf chymosin(rennin) (EC 3.4.23.4) has been determined. The se-quence consists of a single peptide chain of 323 aminoacid residues. The primary structure of the precursorpart of cal f prochymosin was published previously(Pedersen, V. B., and Foltmann, B. (1975) Eur. J. Bio-them. 55, 95-103), thus we are now able to account forthe total 365 amino acid residues of calf prochymosin.Comparison of the sequence of cal f prochymosin withthat of pig pepsinogen A (EC 3.4.23.1) shows extensivehomology. In the precursor part of the sequence, 15residues are located at identical positions, as comparedto 189 identical residues in the respective enzymes.Furthermore comparison to Penicillium janthinellumacid proteinase (penicillopepsin) (EC 3.4.23.7) showsthat 76 residues are common to this enzyme and to thetwo gastric proteinases. These homologies in sequencefurther suggest that the folding of the peptide chain inchymosin is very similar to that of other acid protein-ases.

Chymosin (rennin) (EC 3.4.23.4) is the predominant milk-clotting acid proteinase in the fourth stomach of the cal f, andthe main proteolytic enzyme in cal f cheese rennet. Like pepsinit is secreted as an inactive precursor which is irreversiblyconverted into active enzyme through limited proteolysis re-sulting in a final loss of the 42 NHz-terminal residues of thezymogen ( 1).The general biochemistry of prochymosin and chymosinwas reviewed in 1966 (2). The enzyme may be regarded as arepresentative of one of the major types of gastric proteases.Furthermore x-ray crystallographers are working on the ter-tiary structure (3, 4) and consequently we considered it im-portant to determine the primary structure of chymosin.The fir st experiments were carried out with crystall inechymosin. These resulted in determinations of the sequencearound the S-S bridges, at the NH? terminus and at theCOOH terminus (5-7). The results indicated a high degree ofhomology between the primary structures of chymosin andpepsin (8).By chromatography on DEAE-cellulose the chymosin usedin our experiments could be separated into two main compo-nents (chymosin A and B) (2). The final determination of theprimary structure was performed with chromatographicallypurified preparations. In chymosin B all 323 residues were

* The work was supported by Grants 51 1/674,2601,3581, and 5159from the Danish Natural Scienc e Resea rch Coun cil and by grantsfrom the Carlsberg Foundation. The costs of publication of this articlewere defrayed in part by the payment of page charges. This articlemust therefore be hereby marked aduertisement in accordanc ewith 1 8 USC. Section 1734 solely to indicate this fact.$ Present address, Department of Oral Biology, Scho ol of Den-tistry, University of Wash ington, Seattle, Wash ington 98195.

5 Present address, Zoophy siological Laboratory C, University ofCopenhagen, DK-2100 Copenhagen 0, Denmark.

identified. Of 156 residues identified in chymosin A, only onewas different in chymosin B. A preliminary report on thechymosin sequence has been published (9). This also includesthe NH2-terminal sequence of prochymosin (10).The strategy we followed was conventional and started withthe purification of fragments, either after cleavage by cyano-gen bromide, or after tryptic digestion of amino-blocked en-zyme. The large fragments prepared this way contained from56 to 146 residues. After subsequent proteolytic degradationswe were able to deduce an unambiguous structure, althoughin many experiments we did not recover all peptides. In themain text we describe an outline of the pertinent evidence.Details of materials and methods as well as results are givenin the miniprint supplement.While this work was in progress the amino acid sequence ofporcine pepsinogen A was completed (11-14). Alignment ofcalf prochymosin against porcine pepsinogen A shows that204 amino acid residues are common to the two zymogens.

EXPERIMENTAL PROCEDURESFragmentation of the Peptide Chain-Large fragments were ob-tained by cyanogen bromide cleavage (15, 16) or by tryptic digestion

of protein in which amino groups were blocked by maleylation (17) orcitraconylation (18). For such tryptic digestion we found a greatimprovement in spec ificity if the digestion was carried out at 12C(10, 19). Although in some digestions we obtained only partial cleav-age after arginine 203, well-defined tryptic fragments were obtainedafter digestion at 12C.

Subd igestions were performed with the following enzymes: chy-motrypsin, elastase , Staphyloc occus aureus proteinase, thermolysin,and, after deblocking, trypsin or Armillaria mellea proteinase. Thelatter enzyme cleaves peptide bonds on the amino side of lysineresidues (20). As described in the legend to Fig. 1, peptides aredesignated with letters indicating the digestion s plus the number ofthe first and the last residue.Purification of Peptides-Cyanogen bromide fragments were pu-rified by gel fdtration on Sephadex G-100 in 25% acetic a cid. Forpurification of other aggregating fragments we used gel filtration onSephadex G-100 in 0.05 M NH,HCOl con taining 8 M urea. The firststep of fractionation of subd igests was in most case s gel filtration involatile buffers such as 0.05 M NH,HCO,. The cond itions of theindividual separations are given in legends to the figures.

Final purification of low molecular weight peptides was generallyobtained by high voltage paper electrophoresis or paper chromatog-raphy or both. Diagonal electrophoretic methods were preferentiallyused for purification of peptides containing cystine (21), methionine(22), or lysine (17).

Sequenc e Determination-All Edman degradations were per-formed manually. Carboxypeptidase A (23) and carboxypeptidase Y(24) were used for determinations of some COOH-terminal sequen ces.

In the sequen ce reported in Ref. 9, Fig. 2, misprints have oc curredat position s 160 and 239.* Portions of this paper (including Figs. 4 to 23 and Tab les 1 to 17)are presented in miniprint at the end of this paper. Miniprint is easilyread with the aid of a standard magnifying glass. Full-size photoco piesare available from the Journal of Biolog ical Chemistry, 9650 RockvillePike, Bethesda, Md. 20014. Request Document No. 79M-99, c iteauthor(s), and include a check or money order for $8.10 per set ofphotocopies.

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8448 Primary Structure of Chymosin46 48 50 52 54 56 58 60 62 64 66 68Gly-Glu-Val-Ala-Ser-V~l-Pro-Leu-Thr-Asn-~r-Leu-Asp-Ser-Gln-~r-Phe-Gly-Lys-Ile-~r-Leu-Gly-Thr-Pro-

70 72 74 76 78 80 82 84 86 80 90 1 92 94Pro-Gln-Glu-Phe-Thr-Val-Leu-Phe-Asp-Thr-Gly-Ser-Ser-Asp-Phe-Trp-Val-Pro-Ser-Ile-Tyr-Cys-Lys-Ser-Asn-c>- L%----------J L c , I c , IC IT>- - - - ________________________________________--------------------------------- JE>---- - - -- - -- - -- - -- J L~h~~~ ~~~~~ ~~~~~~ ~~~~~ ~~ Th

196 98 100 102 104 106 108 110 112 114 116 118Ala-Cys-Lys-Asn-His-Gln-Arg-Phe-Asp-Pro-Arg-Lys-Ser-Ser-Thr-Phe-Gln-Asn-Leu-Gly-Lys-Pro-Leu-Ser-Ile-ICI I c , I c

LT------------J cm

Th)-------------------------J 1 Th

, , c I ICCI JT , , CB-T ) CR-T\TM-CB ) TM-CB

I

120 122 124 126 128 130 132 134 136 138 140 142 144His-Tyr-Gly-Thr-Gly-Ser-Met-Gln-Gly-Ile-Leu-Gly-~r-Asp-Thr-Val-Thr-Val-Ser-Asn-Ile-Val-Asp-Ile-Gln-cw ' CB-C II CB 127-169 Edman depradation

CB-TF ,I CB-S , I sTM-CB\-

cs I

) CR"SS

'SS146 148 150 152 154 156 158 160 162 164 166 168Gln-Thr-Val-Gly-Leu-Ser-Thr-Gln-Glu-Pro-Gly-Asp-Val-Phe-Thr-~r-Ala-Glu-Phe-Asp-Gly-Ile-Leu-Gly-Met-

IZ:bJ

J \C 1I I I CB-S J

S> J , s )S

170 172 174 176 178 180 182 184 186 188 190 192 194Ala-Tyr-Pro-Ser-Leu-Ala-Ser-Glu-~r-Ser-Ile-Pro-Val-Phe-Asp-Asn-Met-Met-Asn-Arg-His-Leu-Val-Ala-Gln-CB

S TM

196 198 200 202 204 206 207 209 210 212 214 216 218 220ASp-Leu-Phe-Ser-Val-Tyr-Met-Asp-Asp-Arg-Asp-Gly-Gln-Gl~-S~~-M~t-L~~-Th~-L~~-Gly-Al~-~l~-A~p-p~~~S~~~Ty~~CB>-1 CB I I CB 211-702 Edman degradationTM) II TM-C J 1-i :) A-C 1

FIG. 1. The amino acid sequen ce of chymos in B with sum- indicate end of sequen ce obtained by Edman degradations of themary of the data from which the sequen ce was derived. The entire fragments). Peptides from subd igestions are marked: C, chy-numbering starts from the NH2 terminus of prochymosin. The peptide motrypsin; E, elastase ; S, Staphyloco ccus aureus proteinase; T, tryp-chain of chymosin starts at residue 45. Pos itions 208, 276, 338, 339, sin after deblocking; T/z, thermolysin; A, Armillaria mellea protein-340, and 343 are gaps relative to the peptide chain of porcine pepsin. ase; P, pepsin. Peptides are designated with more than one letterResu lts p resented in this paper are marked by thick lines for residues whenever a series of digestion s, with purification after each step hasidentified by seque ncing and by thin lines for amino a cid compo si- been esse ntial for obtaining the peptide in question; e.g. A-C-S indi-tions. Dashed lines indicate previously published sequen ce da ta (5- cates digestion with A. mellea proteinase followed by digestion with7). The arrows indicate continua tion of peptides from line to line. chymotrypsin and finally with S. aureus proteinase. If the spec ificityThe letters indicate the type of fragmentations from wh ich the of a preceding degradation determines the terminal amino acid residuepeptides were obtained. TM, tryptic digestion of an amino-blocked of a given peptide, such degradations are also indicated. Disulfidepreparation; CB, cleavage with cyanogen bromide (the half-arrows bridges connec t Cys 91 and Cys 96, Cys 252 and Cys 256, and Cys 296terminating the lines of CB fragments at residues 147, 226, and 325 and Cys 329.

RESULTSThe total sequence of chymosin B is presented in Fig. 1together with a summary of evidence from which the sequencewas derived. Previously published sequences (5, 6) that areimportant for the deduction are included in Fig. 1. To facilitatethe comparison between the amino acid sequences of the

gastric proteinases and of their zymogens we use a numberingof residues starting with the NH? terminus of the longest ofthe peptide chains (prochymosin) and count gaps where suchoccur in chymosin relative to pepsin. The numbering of amino

acid residues in porcine pepsin A (13) may be converted toour zymogen numbering by adding 46. With the zymogennumbering the peptide chain of chymosin presented in thispaper starts at residue 45.The thermolytic peptides were obtained from a digest ofintact chymosin. All other peptides were prepared after frag-mentation with cyanogen bromide or digestion with tryps in.Cyanogen Bromide Fragments-Fig. 2 shows a flow dia-gram for purification of the cyanogen bromide fragments.After preliminary experiments with reduced and carboxy-

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Primary Structure of Chymosin222 224 226 228 230 232 234 236 236 240 242 244Tyr-Thr-Gly-Ser-Leu-His-Trp-Val-Pro-Val-Thr-Val-Gln-Gln-Tyr-Trp-Gln-Phe-Thr-Val-Asp-Ser-Val-Thr-Il~-

a449

CB, IL) CB-EC)J A-C ,I A-C A-CI A-C , &-C-S - - ) A-C-S, Th I 1 Th 4

246 248 250 2;2 254 2;6 258 260 262 264 266 268 270Ser-Gly-Val-Val-Val-Ala-Cys-Glu-Gly-Gly-Cys-Gln-Ala-Ile-Leu-Asp-Thr-Gly-Thr-Ser-Lys-Leu-Val-Gly-Pro-CB-E>A CCB-S ,, CB-S ) CB-S

A-C> ,,A )A,T , I T , I T TA-C-S-- I CB-E ) CB-E

L~_______-----------------J TOT CYS272 274 275 277 278 280 282 284 286 288 290 292 294 2% 329

Ser-Ser-Asp-Ile-Leu-Asn-Ile-Gln-Gln-Ala-Ile-Gly-Ala-Thr-Gln-Asn-Gln-Tyr-Gly-Glu-Phe-Asp-Ile-Asp-Cys-CB-S>A , CB-S ,,CB-S > CB-S

A -+AT- , A-E ) A-ECB-E) 1

298 300 302 304 306 308 310 312 314 316 318 320Asp-Asn-Leu-Ser-~r-Met-Pro-Thr-Val-Val-Phe-Glu-Ile-Asn-Gly-Lys-Met-~r-Pro-Leu-Thr-Pra-Ser-Ala-Ty~-CB-S>A 1 CB 11 CB 714-773 Edman denradation ) CB

IAITP

AI

, Th ITOCYS296 322 324 326 1328 330 332 334 336 337 341 342 344 346 348 350Thr-Ser-Gln-Asp-Gln-Gly-Phe-Cys-Thr-Ser-Gly-Phe-Gln-Ser-Glu-Asn-His-Ser-Gln-Lys-Trp-Ile-Leu-Gly-Asp-

CB-A-'*,, T I 1 LT-------------> T

LC_____---_-___---_----------------------------J IC )C\S I

352 354 356 358 360 362 364 366 368 370 372Val-Phe-Ile-Arg-Glu-Tyr-Tyr-Tyr-Ser-Val-Phe-Asp-Ar~-Ala-Asn-Asn-Leu-Val-Gly-Leu-Ala-Lys-Ala-IleT>-----------v-l CTM ,, TM Icw ' TM-C ' , CB-T IILC_---------------A LC-------------------A L-------v---JFig. 1 contd

methylated or reduced and aminoethylated chymosin, i t wasfound that fragments could be more easily purified if thecyanogen bromide cleavage was performed prior to reduction.After the fist gel filtrat ion (Fig. 5), CB 45-126 was obtainedpure, and the low molecular weight peptides were isolated bypaper electrophoresis. CB 211-302 and CB 314-373 were pre-pared after reduction, aminoethylation, and repeated gel fil -trations (Figs. 6 and 7).Tryptic Fragments-From the primary structure shown inFig. 1 it can be seen that cleavage of S-S bridges does notalter the peptides produced from the tryp tic digestion ofmaleylated or citraconylated chymosin. However, reductionof S-S bridges followed by modification of the -SH groupsdid influence the behavior of the fragments in gel filtra tion.Therefore, in most experiments chymosin was first reduced,aminoethylated, and then maleylated before digestion withtrypsin. The purification of the tryptic fragments is summa-rized in Fig. 3, and the corresponding gel filtrat ions are listed.In these experiments, gel filtrat ions gave satisfactory separa-tion of the three large TM fragments.The low molecular weight tryptic peptides were purified bypaper electrophoresis. However, TM 190-203 could not be

purified by paper electrophoresis. It was eventually purifiedby elution of the application zone followed by gel filtrat ion onSephadex G-25.Among the large TM fragments TM 204-354 has a size of asmall protein. The subsequent cleavages were accomplishedwith a combination of degradation procedures: a cleavagewith cyanogen bromide and subsequent digestion withstaphylococcal proteinase; b) deblocking and tryptic digestion;c) digestion with chymotrypsin; d) deblocking and digestionwith A. mellea proteinase followed by digestion sither withchymotryps in and then with staphylococcal proteinase, ordigestion with elastase; e) digestion with staphylococcal pro-teinase.From the sequences of the peptides obtained in the twomajor cleavages (Figs. 2 and 3), we are able to construct anunambiguous complete sequence of chymosin as shown in Fig.1.

DISCUSSION

Comments on the experimental methods and possible ge-netic var iants other than chymosin A and B are given in the


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8450 Primary Structure of Chymosin

FIG. 2. Flow diagram for purifi- reduction and aminoethylatlo,cation of fragments obtained after gel flltratlon sepila*ex G-100 1cleavage with cyanogen bromide. (fig. 6

\1 I I ,

FIG. 3. Flow diagram for purifi-cation of tryptic fragments fromNHz-blocked chymos in followed bycyanogen bromide cleavage of thetwo largest fragments.

last sections of the miniprint supplement.This discussion will be limited to a comparison between thestructure of chymosin and those of other acid proteinases; ofthese, porcine pepsin and penicillopepsin (25) have been ful lysequenced. Alignment shows that of 323 residues in chymosin,189 are identical with the corresponding residues in porcinepepsin. Comparison between chymosin and penicillopepsingives 89 common residues, porcine pepsin and penicillopepsinhave 98 residues in common (25). Of the common residues, 76are found at the same positions in all three enzymes. Thehomology in primary structures suggest a similar folding ofthe peptide chains of these enzymes. This assumption hasbeen sustained by x-ray crystallography of the crystallineenzymes.The tertiary structure of porcine pepsin has been deter-mined by Andreeva et al. (26), and the structure of penicillo-pepsin has been determined by Hsu et al. (25). Two other acidproteinases from Rhizopus chinensis and from Endothiapar-asitica have also been analyzed by x-ray crys tallography.Their primary structures are not yet completed, but the

electron density maps show that they are closely related toeach other (27) and to penicillopepsin (28). Furthermore datafrom x-ray crystallography of chymosin have been comparedto the structure of E. parasitica proteinase. The results o f therotation function provide strong evidence fo r a homologybetween the two enzymes (4), and visual comparison betweenthe tertiary structure of pepsin and the microbial proteinasesalso indicates homology between these enzymes.From these investigations the following general structureappears: the molecules of the acid proteinases are bilobal withan extended hydrophobic interior. The two lobes are sepa-rated by a deep cle ft. Behind the c lef t there is a stack of mixedP-sheet, and the covalent connection between the two lobes isestablished by a strand running on the back of the P-sheet.Only a few turns of cY-helices are found.We are now able to see relationships between the tertiarystructure, the highly homologous sections of the primarystructures and the residues that are identified as catalytica llyactive by inhibition studies. The enzymes are inhibited if oneof two aspartic acid residues are esterified. Aspartic acid 78

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Primary Structure of Chymosin 8451may be esterified with substrate-like epoxides (29, 30), whileaspartic acid 261 reacts with diazo compounds (31, 32). Bothof these groups are located in P-bends pointing toward thecleft o f the molecule, and each bend is crossed by a strandwhich gives rise to I shaped structures (28). With the num-bering used in Fig. 1 the residues of these structures are 77 to81, crossed by 164 to 170, and 259 to 264, crossed by 347 to352. Of the 24 residues in these four sections, 19 are identicalin all three enzymes. This high degree o f identity stronglysupports the view that the catalytic apparatus is the same.James et al. (33) have suggested that tyrosine 121 also partic-ipates in the catalytic mechanism, and again we observe 6identical residues from residue 118 to 125.

Hayashi, Dr. G. Drapeau, and Dr. D. Smyth, respectively. Theinvestigations on chymosin A were mainly carried out by Dr. H.Jacobs en. Mrs. Inge Hgegh, and Mr. E. Aa. Geertsen have contributedwith valuable techn ical assistan ce. We thank a ll for their collabora-tion.

REFERENCES1.

2.3.

Pedersen, V. B., Christensen, K. A., and Foltmann, B. (1979) Eur.J. Biochem . 94,573-580

Foltmann, B. (1966) C. R. Trau. Lab. Carlsberg 35, 143-231Bunn, C. W., Camerman, N., Liang Tung Tsa i, Moews, I. C., andBaumber, M. E. (1970) Philos. Trans. R. Sot. Lond. Ser. B.Biol. Sci. 257, 153-158

4.Other highly homologous sections are found in the regionsthat connect the two lobes. Extensive hydrophobic interac-tions are found in the antiparallel pleated sheet formed byresidue 196 to 200 and 356 to 361. In these sections of thepeptide chain there is only one substitution between pepsinand chymosin and 7 residues are common to all three enzymes.The covalent connection between the two lobes goes fromglycine 214 to leucine 225; here 9 residues are common.

5.6.7.8.9.

Jenkins, J., Tickle, I., Sewe ll, T ., Ungaretti, L., Wollmer, A., andBlunde ll, T. (1977) in Acid Proteases, Structure, Function andBiology (Tang, J., ed) pp. 43-60, Plenum Press, New York

Foltmann, B., and Hartley, B. S. (1967) Biochem. J. 104, 1064-1074

Pedersen, V. B., and Foltmann, B. (1973) FEBS Lett. 35, 250-254Foltmann, B. (1970) Philos. Trans. R. Sot. Lond. Ser. B. Biol.

Sci. 257, 147-151

The eight sections mentioned here comprise 53 residues, ofwhich 41 are common to all three enzymes. The rest of theidentical residues are more evenly distributed, and the signif-icance of these are not obvious with the present information.

Tang, J., and Hartley, B. S. (1970) Biochem . J. 118, 611-623Foltmann, B., Pedersen, V. B., Jacob sen, H., Kauffman, D., and

Wybrandt, G. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2321-232410. Pedersen, V. B., and Foltmann, B. (1975) Eur. J. Biochem . 55,95-103

With the present knowledge we are not able to comment onthe high milk-clotting and low proteolyt ic act ivi ty of chymosinas compared to pepsin. Small differences in the binding sitesof the cleft may be responsible. However, if we assume thatthe tertiary structure of chymosin corresponds to that of otheracid proteinases, we may have an explanation for the differ-ence in milk-clotting act ivi ty between chymosin A and B (2).The only difference in structure that has been found so far isthat residue 290 is aspartic acid in chymosin A and glycine inchymosin B. In the tertiary structure this residue is located inthe opening of the substrate binding cle ft; this will most likelyinfluence the binding of substrate.

11.12.13.

Ong, E. B., and Perlmann, G. E. (1968) J. Biol. Chem. 243,6104-6109

14.15.16.

Pedersen, V. B., and Foltmann, B. (1973) FEBS Lett. 35,255-256Tang, J., Sepulveda, P., Marciniszyn, J., Jr., Chen, K . C. S.,

Huang, W-Y., Tao, N ., Liu, D., and Lanier, J. P. (1973) Proc.Natl. Acad. Sci. U . S. A. 70, 3437-3439Mor&ek, L., and Kostka, V. (1974) FEBS Lett. 43, 207-211Gross, E., and Witkop, B. (1962) J. Biol. Chem. 237, 1856-1860Steers, E., Jr., Craven, G. R., and Anfinsen, C. B. (1965) J. Biol.Chem. 240,2478-2484

17.18.19.20.

Butler, P. J. G., Harris, J. I., Hartley, B. S., and Leberman, R.(1969) Biochem . J. 112,679-689

Gibbons, I., and Perham, R. N. (1970) Bioch em. J. 116,843-849Hapner, K. D., and Wilcox, P. E:. (1970) Biochemistry 9, 4470-4480In addition to porcine pepsinogen and calf prochymosin,fragmentary information is available for another seven verte-brate pepsinogens and pepsins (34). Among these, the NH2-

terminal sequence of bovine pepsinogen A is the longestcoherent sequence published. Out of 110 amino acid residues,84 or 76% are common to bovine and porcine pepsinogen,while only 57 residues or 52% are common to bovine pepsin-ogen and cal f prochymosin. Similarities and differences in theprimary structures are reflected in the immunological reac-tions. When tested against monospecific rabbit antisera, bo-vine pepsin and calf chymosin show no cross-reaction (35),but porcine and bovine pepsin exhibit cross-react ivity. It hasrecently been found that monospecific antisera raised againstcal f chymosin precipitate a proteinase from the stomach o fnewborn pigs, while antisera against porcine pepsin do notprecipitate the proteinase from newborn pigs. This proteinasehas the same optimum for proteolytic activ ity as calf chymosin(pH 3.5) and the name pig chymosin was suggested (36).

Lewis, W. G., Basford, J. M., and Walton, P. L. (1978) Bioch im.Biophys. Acta 522, 551-560

21.22.23.24.25.

Brown, J. R., and Hartley, B. S. (1966) Bioche m. J. 101, 214-228Tang, J., and Hartley, B. S. (1967) Biochem . J. 102, 593-599Ambler, R. I. (1967) Methods Enzymol. 11, 436-445Hayashi, R., Moore, S., and Stein, W . H. (1973) J. Biol. Chem.

248, 2296-2302Hsu, I-N., Delbaere, L. T. J., Jame s, M. N. G., and Hofmann, T.

(1977) Nature 266, 140-14526. Andreeva, N. S., Fedorov, A. A., Gutschina, A. E., Riskulov, R.

R., Shutzkever, N. E., and Safro, M. G. (1978) M ol. Bio l.(Moscow) 12,922-935

27. Subramanian, E., Swan, I. D. A., Liu, M., Davies, D. R., Jenkins,J. A., Tickle, I. J., and Blunde ll, T. L. (1977) Proc. Natl. Acad.Sci. U. S. A. 74, 556-559

28. Tang, J., Jame s, M. N. G., Hsu, I-N., Jenkins, J. A., and Blundell,T. L. (1978) Nature 271. 618-62129.3031

Tang, J. (1971) J. Biol. &em. 246,4510-4517Chen, K. C. S., and Tang, J. (1972) J. Biol. Chem. 247,2566-2574Rajagopalan, T. G., Stein, W. H., and Moore, S. (1966) J. Biol.

Chem. 241, 4295-4297At the moment it is premature to attempt a detailed dis-cussion on the phylogenetic relationships among the acidproteinases, but the results suggest that the chymosins rep-resent a group of neonatal proteinases that have evolvedindependently of the pepsins before divergence of the linesleading to cow and pig.

32 Knowles, J. R., and Wybrandt, G. B. (1968) FEBS Lett. 1, 211-21233. Jame s, M. N. G., Hsu, I-N., and Delbaere, L. (1977) Nature 267,

808-81334. Foltmann, B., and Pedersen, V. B. (1977) in Acid Proteases,

Structure, Function and Biology (Tang, J., ed) pp. 3-22,Plenum Press, New York

Acknowledgments-Carboxypeptidase Y, Staphyloco ccus aureusproteinase, and Armillaria mellea proteinase were gifts from Dr. R.35. Rothe, G. A. L., Axelsen, N. H., Jtlhnk, P., and Foltmann, B.

(1976) J. Dairy Res. 43, 85 ?536. Foltmann, B., Lenblad, I-., and Axelsen, N. H. (1978) Biochem. J.169,425-427

N. H. Axelsen, personal comm unication. Additiona l references are found on p. 8456.


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Primary Structure of ChymosinTszIL

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a454 Primary Structure of Chymosin

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