Vol. 7I ORIGIN OF OAK-BARK TANNIN 537the period of active photosynthesis and growth,and the enhanced level is maintained until thefollowing spring. In the older trces, this pro-gressive increase in tannin content with increase inage is balanced by increase in the dead and livingbark elements, with the result that the levelremains without apparent variation throughout theseason. This suggests that the taImin undergoesfurther chemical changes in the dead elements ofthe outer bark.

3. The effect of ringing experiments on thenormal distribution of tannin in the stembark ofmature trees shows an accumulation of tanninabove the girdle, suggesting that synthesis hastaken place from phenolic precursors which arenormally translocated downwards from the leaves.

4. Examination of the exudate obtained bytapping the sieve-tube system of growing treesshows the presence there of many of the phenolicsfound in the bark, including the pyrogallol pre-cursors [(+ )-gallocatechin and leucodelphinidin] ofoak-bark tannin.

5. The evidence suggests that these pyrogallolphenols, which originate in the leaves, are trans-located by the sieve-tube system to the cambiumand undergo oxidation there, and the resultingphlobatannin is stored in the bark.

The author thanks the Director and Council ofthe BritishLeather Manufacturers' Research Association for permis-sion to publish this paper.We are also indebted to the Forestry Commission for

providing facilities, and to Mr G. D. Holmes, Silviculturistto the Forestry Commission, of Alice Holt Lodge ResearchStation, Wrecclesham, Hants, and Mr J. R. Aaron,Development Officer to the Forestry Commission, of theDepartment of Research and Education, Savile Row,London, W. 1, for making the arrangements for the field

experiments. We thank Messrs J. J. Bennison, R. D. G.Lane and J. J. C. Peddie for technical assistance.

REFERENCES

Clark, R. H. & Andrews, H. I. (1921). Industr.EngngChem.(Industr.) 13, 1026.

Cromwell, B. J. (1933). Biochem. J. 27, 860.Grassmann, W., Endisch, 0. & Kuntara, W. (1951). Das

Leder, 2, 202.Grassmann, W. & Kuntara, W. (1941). Collegium, Haltigen,

855, 187.Hartig, T. (1860). Allg. Fordt- u. Jagdztg. 36, 257.Hathway, D. E. (1958). Biochem. J. 70, 34.Kennedy, J. S. & Mittler, T. E. (1953). Nature, Lond., 171,

528.Mason, T. G. & Maskell, E. J. (1928). Ann. Bot., Lond.,

42, 1.Mason, T. G. & Maskell, E. J. (1929). Ann. Bot., Loud., 43,

205.Mason, T. G. & Maskell, E. J. (1934). Ann. Bot., Lond., 48,

119.Mittler, T. E. (1953). Nature, Lond., 172, 207.Mittler, T. E. (1958). In The Physiology of Forest Trees,

p. 401. Ed. by Thimann, K. V. New York: The RonaldPress Co.

Munch, E. (1930). In Die Stoffbewegungen in der Pflanze,pp. 1-234. Jena: Gustav Fischer.

Official Methods of Analysis (1957), p. 14. Croydon: TheSociety of Leather Trades' Chemists.

Rogers, J. S., Calderwood, H. N. & Beebe, C. W. (1950).J. Amer. Leath. Chem. Ass. 45, 733.

Thomas, M., Ranson, S. L. & Richardson, J. A. (1956). InPlant Physiology, 4th ed., p. 181. London: J. and A.Churchill.

Ziegler, H. (1956). Planta, 47, 447.Zimmermann, M. H. (1957). Plant Physiol. 32, 288.Zimmermann, M. H. (1958). In The Physiology of Forest

Trees, p. 381. Ed. by Thimann, K. V. New York: TheRonald Press Co.

Substrate Specificity of Phosphoprotein Phosphatase from Spleen

BY T. A. SUNDARARAJAN AND P. S. SARMAUniversity Biochemical Laboratory, Madra8-25, India

(Received 17 July 1958)

Recent investigations from several Laboratorieshave revealed the presence, in mammalian tissues,of an enzyme system for the dephosphorylation ofphosphoproteins, which appears to be distinct fromphosphomonoesterasos (Feirstein & Volk, 1949;Norberg, 1950; Mattenheimer, 1953; Thoai, Roche& Pin, 1954; Sundararajan & Sarma, 1954, 1957).Evidence for the specific nature of the enzyme haslargely been based on its inability, to split -glycerophosphate,. a typical phosphomonoestersubstrate. A detailed study of th-e substrate

specificity of this enzyme is warranted in view ofthe recent observation of Perlmann (1955) thatphosphoproteins containing phosphomonoesterlinkages are susceptible to dephosphorylation byphosphomonesterases. The present paper describesthe results of an investigation on the specificity ofhighly purified preparations of phosphoproteinphosphatase from ox spleen towards a number ofsubstrates representing different types of phos-phorus linkages. As a parallel study, the specificityof a partially purified preparation of the enzyme

T. A. SUNDARARAJAN AND P. S. SARMAfrom rat spleen has also been investigated. Thesestudies indicate that the mammalian enzyme

possesses a wide substrate specificity, since itreadily dephosphorylates a variety of compoundscontaining pyrophosphate linkages and alsohydrolyses the phosphomonoesters of phenol andp-nitrophenol.

While the manuscript of this paper was in pre-

paration, the attention of the authors was drawn toa publication of Hofman (1958), who reportedessentially similar results with a partially purifiedenzyme from ox spleen. The present paper providesevidence of a more conclusive nature for theidentical characteristics of the enzyme system de-phosphorylating the various substrates.

MATERIALS AND METHODS

Enzymes. Purification of phosphoprotein phosphatasefrom ox spleen was effected by a modification of the pro-

cedure published earlier (Sundararajan & Sarma, 1954) andthe details are given below. Rat-spleen phosphatase was

prepared according to Sundararajan & Sarma (1957).Protein substrates. Casein was prepared by the method

of Van Slyke & Baker (1918). The preparation of ,-caseinwas carried out according to Hipp, Groves, Custer &MeMeekin (1952), with urea as fractionating agent. Phos-vitin was prepared from egg yolk according to the methodof Mecham & Olcott (1949). Phosphopeptone was pre-

pared from fl-casein by digestion of the protein withcrystalline trypsin (Worthington Biochemical Co., N.J.,U.S.A.) according to the method of Peterson, Nauman &McMeekin (1958). The preparation thus obtained appearedto be electrophoretically homogeneous.

Phosphomonoesters. Substrates employed for the detec-tion of phosphomonoesterase activity were: ,B-glycero-phosphate (British Drug Houses Ltd.); phenyl phosphate(disodium salt), p-nitrophenyl phosphate (disodium salt)and phenolphthalein phosphate (which were generous giftsof Sigma Chemical Co., St Louis, Mo., U.S.A.); O-phos-phorylserine, kindly supplied by California Foundation forBiochemical Research, Los Angeles, U.S.A.; glucose 1-phosphate (dipotassium salt), glucose 6-phosphate (bariumsalt), fructose 6-phosphate (barium salt) and fructose 1:6-diphosphate (barium salt), all purchased from SchwarzLaboratories Inc., New York, U.S.A.

Pyrophosphate esters. Sodium pyrophosphate (BakersAnalysed, J. T. Baker Chemical Co., Phillipsburg, N.J.,U.S.A.) was employed for detection of pyrophosphataseactivity. Adenosine triphosphate (ATP) was obtained asthedisodium salt from Pabst Laboratories, Milwaukee, U.S.A.Thiamine pyrophosphate hydrochloride was a product ofHoffman-La-Roche and Co., Basle, Switzerland.

Phosphoamides. Creatine phosphate and N-phosphoryl-DL-phenylalanine methyl ester were kindly supplied byDr Si-Oh-Li.

Phosphodiesters. Diphenyl phosphate, a generous giftfrom HM Chemical Co., Calif., U.S.A. and p-bis(nitro-phenyl) phosphate (calcium salt), kindly supplied bySigma Chemical Co., were used as phosphodiester substrates.When the barium salts of substrates were used, the

barium was quantitatively removed as barium sulphate.

All substrate solutions were adjusted to the pH of theincubation mixture before use.

Buffer. Sodium acetate-acetic acid buffer (0-05M) was

used throughout the studies (Walpole, 1914).Determination of phosphatase activity. Methods for the

determination of phosphatase activity and definition ofunit of enzyme activity are as described earlier (Sundarara-jan & Sarma, 1954). Phosphorus determinations were

carried out according to the method of Fiske & Subbarow(1925). The method of Lowry & Lopez (1945) was used formeasurement of phosphoamidase activity. Phospho-diesterase activity was determined by estimation of theliberated phenol according to King (1947).

Determination of protein nitrogen. This was determined bydigestion of the protein with H2SO4 and estimating theammonia titrimetrically (Ma & Zuazaga, 1942) or by thenessler reaction (Koch & McMeekin, 1924). The colori-metric method of Lowry, Rosebrough, Farr & Randall(1951) was used for measurement oflow amounts of protein.

Purification of phosphoproteinphosphatase from ox spleen

The method of Sundararajan & Sarma (1954) was

modified in several respects to get preparations of highspecific activity. The use of a buffer at pH 5 for renderingthe inactive proteins of spleen insoluble, and of manganesepyrophosphate gel for adsorption of the enzyme, consti-tuted two of the important steps introduced in the follow-ing modified procedure.

Extraction of the enzyme. A weighed amount of mincedspleen was homogenized with 2-5 vol. of 2-5% (w/v) NaClsoln., buffered at pH 5-0 with 0-2M-acetate buffer. Thehomogenate was centrifuged at 2000 rev./min. for 15 min.and the supernatant was filtered on a Buchner funnel,with 10g. of Celite L665/1. as filter aid. (L665 was obtainedfrom Fisher Scientific Co., U.S.A.) (Table 1, stage 1.)

Fractionation with ammonium sulphate. The clear filtratewas treated with solid (NH4)2SO4 to 0-5 saturation (theamount required for bringing solutions to any desiredsaturation level being calculated from the formula ofKunitz, 1952). After allowing the solution to stand over-

night in the ice chest, it was centrifuged and the super-natant filtered through a layer ofCelite on a Buchner funnel.The clear amber-coloured filtrate was treated with more

(NH4)2SO4 to 0-85 saturation. The solution, after storage at0° overnight, was filtered on a large Buchner funnel. Whenthe filter cake had been sucked almost dry, it was removedfrom the paper, suspended in water and dialysed at 50against several changes of distilled water. After dialysis theprecipitated protein was separated by centrifuging andextracted with 2-5% (w/v) NaCl soln. at pH 5-0 (stage 2).

Adsorption on manganese pyrophosphate. The clearextract obtained from the previous step was adsorbed on

manganese pyrophosphate gel. The adsorbent was preparedby the gradual addition of 0-1M-sodium pyrophosphate to2 vol. of 0- 1 M-manganous sulphate which was kept wellstirred during the addition. The ppt. was allowed to settleand the supernatant removed by decantation. The gel wasrepeatedly washed with 1% (w/v) NaCl soln. and finallyfiltered on a Buchner funnel without applying suction. Theppt. was removed and suspended uniformly in water. Onlyfreshly prepared gels were used since those allowed to ageat room temperature or in the ice chest were inefficient asadsorbents. For adsorption studies, 1 vol. of the enzyme

I959538

SPECIFICITY OF PHOSPHOPROTEIN PHOSPHATASEsolution containing about 10 mg.-of protein/ml. was mixedwith an equal volume of the gel (6x5 mg. of total solids/ml.).Enough water was added to bring down the concentrationof NaCl to 1%. The suspension was stirred for 2 min. andcentrifuged. The gel was washed once with 1 % NaCl atpH 5. Elution of the enzyme was carried out by taking upthe residue in 0-2 saturated (NH4)2SO4, and after gentlestirring for 5 min. at room temperature the suspension wascentrifuged (stage 3).

Refractionation with ammonium sulphate. The eluatefrom manganese pyrophosphate gel was subjected torefractionation with (NH4)2804. The ppt. separatingbetween 0-58 and 0-85 saturation was collected by centri-fuging and was extracted with NaCl soln. (stage 4).

Fractionation with acetone. The extract from the previousstep was subjected to fractionation with acetone, accordingto the procedure outlined earlier (Sundararajan & Sarma,1954). The active fraction, separating between 50 and 66%(v/v) concentration of acetone, was separated and extractedwith saline (stage 5).

The purification achieved in a typical fractionationexperiment is outlined in Table 1. The final fraction, whichrepresented a 600-fold purification over the initial spleenextract, was used as source of enzyme in specificity studies.

RESULTS

Sub8trate specificity of phosphoproteinphosphatase

The action of purified preparations of the enzymefrom spleen on several phosphate esters is sum-marized in Table 2. The action of the enzyme oneach substrate was studied at various pH valuesranging from 4 to 6. The values given in the tablerepresent the hydrolysis observed at the optimumpH for each substrate.

Dephosphorylation of phosphomonoesters. Ali-phatic phosphate esters were not hydrolysed to any

Table 1. Purification of phosphoprotein phosphatase from ox spleenActivities were tested with casein (5pg.atoms of P/ml.) as substrate and thioglycollic acid (mM) as activator. Incuba-

tions were carried out at 370 and at pH 5-8 for a period of 15 min. Details of stages of purification are given in the text.Specific

Stage Volume Activity Protein N activity Yieldno. (mi.) (units/ml.) (mg./ml.) (units/mg. of N) (%)1 1790 14-7 4 30 3-4 1002 60 322 1-34 240 743 80 140 0-14 1000 434 20 224 0-14 1600 175 20 217 0.11 2000 16

Table 2. Substrate specificity of spleen-phosphoprotein phosphataseThe substrates, at the indicated concentrations, were incubated with the enzyme preparation for 60 min. at 370 with

thioglycollic acid (mM) as activator. Activities were tested at pH 5-8 for casein and at pH 5 0 for the other substrates,except that with ox-spleen phosphatase acting on phenyl phosphate, the pH of the medium was 4-5.

SubstrateCaseinPhosphomonoesters

fl-GlycerophosphateO-PhosphorylserineGlucose 1-phosphateGlucose 6-phosphateFructose 1-phosphateFructose 1:6-diphosphatePhenyl phosphatep-Nitrophenyl phosphatePhenolphthalein phosphate

PhosphoamidesN-PhosphorylphenylalaninePhosphocreatine

Pyrophosphates and phosphodiesterInorganic pyrophosphateAdenosine triphosphateThiamine pyrophosphateDiphenyl phosphate

Concn. ofsubstrate

(ILg.atoms ofP/mi.)5.0

5.05-02-52-52-52-55-05.05.0

1*61-6

Phosphorus released(pg.atoms of P/ml.)

Rat-spleen Ox-spleenphosphatase phosphatase

1-75 1*25

NilNil0-010-020-030-061-561-760.09

NilNilNil0-020-010-021-431-930-09

0-530-13

2-5 2-03 2-102.5t 2-28 2-202-5 * 1-202-5 Nil Nil

*entration given corresponds to acid-labile phosphorus.

VoI. 7I 539

t Concl* Not tested.

T. A. SUNDARARAJAN AND P. S. SARMA

appreciable extent by the enzyme. In marked enzyme preparation. This finding agrees with thatcontrast, the phosphomonoesters of phenol and p- of Singer & Fruton (1957), who demonstrated thenitrophenol were dephosphorylated at a rapid rate. identical nature of phosphoprotein phosphataseMaximum hydrolysis of phenyl phosphate occurred and phosphoamidase of ox spleen.at pH 40-4-5 (Fig. 1), and there was a marked fall Dephosphorylation of pyrophosphate and phos-in activity below pH 4-0. Phenolphthalein phos- phodiester sub8trate8. Compounds containing pyro-phate, though an aromatic ester, was dephos- phosphate linkages appeared to be very good sub-phorylated at a much slower rate. strates for the enzyme. Inorganic pyrophosphate

Dephosphorylation of phosphoamides. N-Phos- and ATP were hydrolysed at comparable rates.phorylphenylalanine and phosphocreatine were de- Both the acid-labile phosphate groups of ATP werephosphorylated to an appreciable extent by the cleaved by the enzyme, whereas the a-phosphoruspurified enzyme from ox spleen, indicating the atom appeared to be resistant to enzymic action.presence of phosphoamidase activity in the This is in conformity with the finding of Hofman

(1958) that ox-spleen phosphatase does not de-phosphorylate adenosine 5'-phosphate. Thiamine

300 pyrophosphate was also readily dephosphorylated,only the terminal phosphate being split off by theaction of the enzyme. Maximum hydrolysis of the

250pyrophosphate substrates was observed at pH 5-0

250 v sX / > (Fig. 1). The enzyme had no action on diphenylphosphate, a diester substrate, even after prolongedincubation.

200 The close similarity in specificity character-/ istics, observed with enzyme preparations of

different states of purity and derived from entirelydifferent sources, suggested that the hydrolysis of

@ 150 _ / \ \ \ the various substrates was mediated by a singleDX 4 \ \ enzyme or by a system of enzymes with similar

properties. This was strikingly borne out by theobservation that the ratio of the activities towards

100 - x the phosphoprotein and pyrophosphate substratesremained more or less constant in the course ofpurification of the enzyme from ox spleen (Table 3).Singer & Fruton (1957) have, by a simnilar criterion,

50 - shown that the phenylphosphatase and phospho-amidase of ox spleen possess properties similar tothose of phosphoprotein phosphatase. Evidencefor the identical nature of the enzymes was also

,3-5 40 4-5 5- 5-5 6-0 6-5 obtained from heat-denaturation and adsorptionpH of the medium experiments as well as from studies on the be-

Fig. 1. Effect ofpH on the dephosphorylation ofATP ( x, haviour of these enzymes towards several acti-pyrophosphate (0) and phenyl phosphate (S) by ox- vators and inhibitors. Details of these studies arespleen phosphoprotein phosphatase. given below.

Table 3. Hydrolysis of casein and pyrophosphates by active fractions froM ox spleen

Active fractions obtained at various steps during purification of phosphoprotein phosphatase were tested for theirability to dephosphorylate casein (20,ug.atoms of protein P/ml.), inorganic pyrophosphate (20,ug.atoms of P/ml.) and ATP(10 ,ug.atoms of acid-labile P/ml.). Incubations were at 370 for 15 min. with thioglycollic acid (mm) as activator. Stages ofpurification of the enzyme are as in Table 1. One unit is the amount ofenzyme catalysing the splitting of 1 Ag. of inorganicP/min. at 370.

Stageno.123,45

Casein14-7

322140224217

Activity (units/ml.)

Pyrophosphate40547420760533 -

ATP23-3

287200200145

Ratios

Casein/pyrophosphate

0-40-60-30-30-4

Casein/ATP0-61-10-71.11-5

5140 II959

SPECIFICITY OF PHOSPHOPROTEIN PHOSPHATASEEffect of heat-treatment on pho8phata8e activity.

Samples of the purified enzyme from ox spleenwere held in a water bath at 800 for various periods.At the end ofthe indicated time interval, the samplewas chilled in ice and the activity estimated withcasein and pyrophosphate as substrates. The loss inactivity towards both the substrates was observedto be of the same order of magnitude (Table 4).The heat-treated preparations were also found tobe active towards ATP and phenyl phosphate.

Adsorption 8tudies. Calcium phosphate gel wasfound to be a poor adsorbent for the enzyme. Thesupernatant solution obtained after treatment withcalcium phosphate exhibited activity against caseinas well as pyrophosphate. Selective adsorption ofphosphoprotein phosphatase on casein was tried bymixing a portion (0-2 ml.) of the enzyme from oxspleen with 2 ml. of 10% (w/v) solution of caseinand precipitating the casein by addition of diluteacid (O lN-HCl) to pH 4-8. The supernatant ob-tained by centrifuging was, however, found to beinactive towards all the four substrates. Similarresults were obtained with manganese pyrophos-phate gel, which adsorbed the protein phosphataseas well as pyrophosphatase. The eluates obtainedafter treatment of such gels with 0-2 saturatedammonium sulphate exhibited activity towardscasein, pyrophosphate, ATP and phenyl phosphate.

Activation and inhibition studiem. Thioglycollicacid at mM-concentration had a marked activatingeffect on the hydrolysis of the various substrates bythe purified enzyme from ox spleen (Table 5).Sodium tungstate at a concentration of lObMcompletely suppressed the hydrolysis of the varioussubstrates. L-Tartrate at a concentration of 10 mmdid not inhibit the hydrolysis of any of thesesubstrates. Incidentally, this observation serves todifferentiate the enzyme from the acid phosphataseof spleen which is sensitive to tartrate (Abul-Fadl&= King, 1949). Enzyme preparations allowed tostand at pH 9-0 at room temperature for a period of1 hr. were completely inactive against all the sub-strates.

Evidence for the participation of a single enzymein the hydrolysis of the various substrates was alsoprovided by the observation that the simultaneouspresence of any two of the substrates in theincubation mixture caused mutual inhibition oftheir enzymic dephosphorylation. This is evidentfrom the results presented in Table 6, which showsthat p-nitrophenyl phosphate inhibits the hydro-lysis of pyrophosphate and of phosvitin in a re-ciprocal manner. The dephosphorylation of the

Table 4. Effect of heat-treatment on phosphoproteinphosphata8e and pyrophosphata8e from ox spleenPortions of the purified enzyme from ox spleen were held

in a water bath at 800 for the indicated periods and theactivity was determined against casein (20pjg.atoms ofP/ml.) and inorganic pyrophosphate (20ILg. atoms ofP/ml.). Incubation was at 370 for 30 min. and at pH 5-8with thioglycollic acid (mM) as activator.

Period ofheating(min.)

051015

Phosphorus released(/Ig.atoms/ml.)

With Withcasein pyrophosphate1-85 2-100-90 1-600-73 0-970-73 0-97

Table 5. Effect of thioglycollic acid on hydrolysis ofvarious substrates by phosphoprotein phosphataseDephosphorylation of the indicated substrates by puri-

fied enzyme preparation from ox spleen was followed inthe presence and in the absence of thioglycollic acid(mM-concn., when present). Incubation was carried out atpH 5-8 for 30 min. at 37°.

P n. a

SubstrateCaseinInorganic pyrophosphateATPPhenyl phosphate

irnospnorus3 rceaaeu(Ag.atoms/ml.)

With Withoutactivator activator

2-0 0-31-6 0-41-5 0-41-3 0-5

Table 6. Mutual inhibition of hydroly8i8 of p-nitrophenyl phosphate, pyrophosphate and of phoavitinPurified enzyme preparation from rat spleen was incubated with the indicated substrates at concentrations correspond-

ing to 5,g.atoms of P/ml. at 370 for 15 min. and at pH 5-0 with thioglycollic acid (mM) as activator.

Phosphorus released (ug.atoms/ml.) from

p-itrphnySubstrate

p-Nitrophenylphosphate Pyrophosphate Phosvitin

p-Nitrophenyl phosphate 0-93Pyrophosphate 1-40Phosvitin - 0-28p-Nitrophenyl phosphate +phosvitin* 0-50 0-10p-Nitrophenyl phosphate + pyrophosphate* 0-68 0-70

* Phosphorus contributed by p-nitrophenyl phosphate was calculated from the amount of p-nitrophenol formed, asestimated according to Sinsheimer & Koerner (1952). This value was deducted from the total phosphorus to give theamount of phosphorus formed from the other substrate.

5;41

T. A. SUNDARARAJAN AND P. S. SARMA

Table 7. Effect of pho8phodie8ter 8ub8trate on hydroly8is of ,-ca8ein by spleen pho8phata8e

The influence of p-bis(nitrophenyl) phosphate on hydrolysis of ,-casein and ,-casein phosphopeptone by rat-spleenphosphatase was studied. The substrates at a concentration corresponding to 1-25 ug.atoms of P/ml. were incubated withthe enzyme at 370 for 15 min. with thioglycoilic acid (mM) as activator. The pH of the medium was 5-8 for casein and 5-0for the peptone.

Substrate)3-Casein,-Casein,-Caseinf-Casein peptoneP-Casein peptone,-Casein peptone*,B-Casein peptone*

Conen. ofdiester

(,ug.atoms of P/ml.)Nil1-2550Nil5-0Nil5-0

Phosphorusreleased

(2g.atom5/ml.)0-250-250-180-130-130-550-55

Inhibition(%)

Nil28

Nil

Nil* Incubation time 1 hr.

Table 8. Hydroly8i8 of pho8phopeptone fromr,B-casein by spleen phosphata8e

Purified enzyme from rat spleen was incubated with thesubstrates at a concentration corresponding to 1-25,ug.-atoms of P/ml. for 60 min. at 370 with thioglycollic acid(mM) as activator. Values for phosvitin are given forpurpose of comparison.

Substrate,-Casein

,-Casein peptone

Phosvitin

pH ofmedium

5-05-84-55-05-84.55-05-8

Dephos-phorylation

(%)

1776

last-named substrates was not affected by thepresence of p-nitrophenol in the medium. Theresults show that these substrates inhibit eachother by competition for the same active centre on

the enzyme surface.

Dephosphorylation of fl-ca8sein by8pleen pho8phata8e*

Perlmann (1954a) has recently achieved a step-wise dephosphorylation of ,B-casein by incubationof the protein with phosphodiesterase from snakevenom followed by treatment with a phospho-monoesterase. In view of the non-specific nature ofspleen phosphatase it was of interest to investigatethe mechanism of dephosphorylation of fl-casein bythis enzyme with reference to the possible partici-pation of a phosphodiesterase in this reaction.Substrate-specificity studies indicated, however,the virtual absence of phosphodiesterase activity in

* These investigations were carried out recently duringthe tenure of a Post-Doctoral National Research Fellowshipawarded to one of us (T. A. S.) by the Ministry of Education,Government of India.

purified phosphatase preparations from spleen(Table 2). Evidence against the participation ofdiesterase in this reaction was also provided by theobservation that the dephosphorylation of p-casein was not appreciably affected by the presenceof a large excess of a phosphodiesterase substrate inthe incubation mixture (Table 7). Similar resultswere obtained when p-casein was replaced by aphosphopeptone from ,-casein which represents,according to Peterson et al. (1958), the phosphorus-containing core of the protein.The action of phosphoprotein phosphatase on

phosphopeptone is shown in Table 8. Earlierexperiments, carried out with non-homogeneouspreparations of the peptone, indicated that thissubstrate was resistant to dephosphorylation atpH 6-0 (Sundararajan & Sarma, 1954). The resultswith P-casein peptone confirm this observation andshow besides that the peptone has a lower pHoptimum as compared with the parent protein. Thevalues obtained with P-casein and its degradationproduct are not strictly comparable in view of thelimited solubility of the protein substrate belowpH 5-5. It was hence considered of interest toinvestigate the true pH optimum for protein-dephosphorylation with phosvitin as substratesince this protein remains soluble over the entirerange of pH used in these studies. From theresults presented in Table 8 it is evident thatphosvitin is more akin to casein than to phospho-peptone in its dephosphorylation behaviour. Thereason for the shift in pH optimum observed withthe peptone is not clear.

DISCUSSION

The results of the present investigation establishbeyond question the non-specific nature of phos-phoprotein phosphatase of mammalian origin.Thus the enzyme preparations exhibit high activityagainst several other substrates besides phospho-protein, and this specificity pattern remains un-

542 I959

Vol. 7I SPECIFICITY OF PHOSPHOPROTEIN PHOSPHATASE 543

altered even after extensive purification of theenzyme. Attempts to eliminate the pyrophos-phatase and phenylphosphatase activity from thepreparation through various techniques haveproved uniformly unsuccessful. The results obtainedin the course of these experiments indicate on thecontrary that the dephosphorylation of the varioussubstrates is mediated by a single enzyme. In viewof these findings the enzyme must be considered tobe distinct from the phosphoprotein phosphatasesof frog eggs and of chick embryo, which appear tobe strictly specific for phosphoprotein substrates(Harris, 1946; Foote & Kind, 1953).The unusual specificity characteristics exhibited

by spleen phosphatase poses a problem as to itsclassification in the scheme of Folley & Kay (1936).There appears to be little justification for classi-fying the enzyme as a phosphomonoesterase sinceit hydrolyses pyrophosphate esters as readily as itdoes phenyl phosphate and, besides, th-e two sub-strates appear to combine with the enzyme at acommon active centre. Further, the enzymediffers from the common acid phosphatases inshowing a high degree of specificity towardsphenyl phosphate among the phosphomonoestersubstrates. Similar results have been reportedrecently by Singer & Fruton (1957) and by Hofman(1958). Judging from the specificity pattern ofspleen phosphatase, it might appear that thephosphorus in phosphoproteins is linked to thetyrosine hydroxyl groups of the protein rather thanto the aliphatic hydroxyl group. However, thepresence of such linkages in casein was ruled outby the observation that the removal of phosphorusfrom the protein did not lead to any increase in thefree phenolic groups of the protein, as tested withphenol reagent (Sundararajan, 1956). The virtualabsence of tyrosine in phosvitin, a protein with thehighest phosphorus content, may also be taken asan indication of the non-involvement of a tyrosinehydroxyl group in the binding of phosphorus inphosphoproteins.The presence, in proteins, of phosphorus linkages

other than the monoester type was first suggestedby Perlmann (1954a) from her experiments on theenzymic dephosphorylation of ,-casein. Thisprotein was dephosphorylated by prostate phos-phatase only after a preliminary treatment of theprotein with phosphodiesterase prepared fromsnake venom. It was hence suggested that thephosphorus in this protein was present exclusivelyas diesterified phosphate. The results of our ex-periments with ,B-casein do not, however, supportthis conclusion (Table 7). The presence of diesterlinkage in casein is made less likely by the obser-vation that phosphatase preparations derived fromother sources and essentially free from diesteraseactivity are also effective in bringing about ex-

tensive dephosphorylation of the protein (Mecham& Olcott, 1949; Sampath Kumar, 1958).The high pyrophosphatase activity exhibited by

spleen phosphatase is of interest in view of therecent report by Perlmann (1954b) on the presenceof pyrophosphate linkage in a-casein. Employingcrystalline yeast pyrophosphatase, she showed thatabout 20% of the phosphorus in a-casein waspresent as pyrophosphate. It is thus possible thatthe rapid dephosphorylation of casein by phos-phoprotein phosphatase depends, in part, on thepyrophosphatase activity inherent in these pre-parations. In view of the non-specific nature ofspleen phosphatase the results of the presentstudies cannot be considered as providing con-clusive proof for the presence of pyrophosphatelinkages in proteins. The use of purified enzymesstrictly specific for phosphomonoester linkage may,however, be expected to throw more light on thepossible occurrence of these types of phosphoruslinkages in proteins. The recent work on the isola-tion and characterization of such enzymes from avariety of sources (Morton, 1955; Tsuboi & Hudson,1955, 1956) appears encouraging in this respect.

SUMMARY1. A method is described for preparing phospho-

protein phosphatase of high specific activity fromox spleen. The enzyme was purified 600-fold witha yield of 16 %.

2. The enzyme was non-specific in its action.A number of substrates containing pyrophosphatelinkage were readily dephosphorylated as alsowere the phosphomonoesters of phenol and p-nitrophenol.

3. Attempts to separate an enzyme specific forphosphoproteins proved unsuccessful. The evidenceobtained indicated that a single enzyme wasresponsible for the dephosphorylation of thevarious substrates.

4. The purified enzyme from spleen had nodiesterase activity and dephosphorylated f-caseinreadily even in the presence of an excess of phos-phodiester. These results suggest the absence ofphosphodiester linkages in P-casein.

5. The significance of these findings in relationto the nature of phosphorus linkages in casein isdiscussed.We wish to thank the University of Madras for permis-

sion to publish these results, which form part of a thesissubmitted by one of us (T. A. S.) for the degree of Doctor ofPhilosophy in January, 1956.

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Nephelometric Determination of Elastase Activity andMethod for Elastoproteolytic Measurements

BY ILONA BANGA, J. BALO AND MAGDOLNA HORVATHThe First Department of Pathological Anatomy and Experimental Cancer Re8earch

Medical Univer8ity, Budape8t

(Received 15 April 1958)

Measurements on the pancreatic elastase activityare performed by different methods in differentlaboratories. Bal6 & Banga (1950) and Banga(1952) suggested a gravimetric method in whichthe fraction of insoluble elastin that has not beendissolved by the enzyme is determined. The samemethod was used by Lewis, Williams & Brink(1956). Hall (1955) and Hall & Gardiner (1955)used the biuret reagent described by Sols (1947) indetermining the elastin dissolved by the elastase.The Folin-phenol reagent (Lowry, Rosebrough,Farr & Randall, 1951) was applied by Grant &Robbins (1957) to measure the proteolytic dissolu-tion of elastin. Sachar, Winter, Sicher & Frankel(1955) measured the colour liberated during theelastolytic action of elastase on elastin which waspreviously stained with orcein. Robert & Samuel(1957) measured the elastolytic dissolution of'azoelastin' by a spectrophotometric method andgave some data for the kinetics of elastolysis.In this paper we describe a method by which the

activity of different elastase preparations obtainedin different laboratories may be compared. The

need for such a method is emphasized by the factthat the mechanism of elastolysis is far from beinguniform and well understood.The enzymic dissolution of elastin, i.e. elasto-

lysis, is not a mere proteolysis. Banga (1951)stressed that no free amino groups are demon-strable by- the formol method or the Van Slykedetermination during the dissolution process. Hall,Reed & Tunbridge (1952) and Hall (1953) believethat a polysaccharide and a sulphate group areliberated. Hall & Gardiner (1955) performedquantitative measurements on the amount ofliberated acid during elastolysis and considered theacid, in accordance with the original assumption ofHall et al. (1952), to be an organically bound sul-phate. Since they held the view that the sulphateion would be bound to mucopolysaccharide, theyconceived the elastolysis to be mucolysis. Partridge& Davis (1955), on the other hand, demonstratedthe formation of free x-amino groups with thefluorodinitrobenzene technique. This led them tosuppose that elastase or one of its components is aproteolytic enzyme. These authors found no more