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THE JOURNAL OF BIOLOGICAL CHEMISTR YVol. 248, No. 5,Issue of March 10, pp. 1741-1745, 1973Printed in U.S .A.

AsparaginaseSTEREOCHEMISTRY OF THE ACTIVE CESTER AS DETERMINED BY CIRCULAR DICHROISXI OFSUBSTRATES*

(Received for publicat,ion, July 7, 1972)

JOSEPH E. COLEMAN AND ROBERT E. ~ANDscHunIAcHERFrom the Departments o;f Molecular Biophysics and Biochemistry and Pharmacology, Yale Universi ty,New Haven, Connecticut 06510

SUMMARYLarge Cotton effect s associated with the r + T* and

n + g* transitions of asparaginase substrates have beenshown in substrates containing a 2nd asymmetrical carbonatom at the p position, diaminosuccinic acid monoamide and@-methylasparagine. Hydro lysis of the amide bond is ac-companied by large changes in the rotatory strength of thesetransitions. These substrates have four possible isomers,L-L, D-D, L-D , and D-L. From the signs of the Cotton effectsappearing on hydrolysis of mixtures of these isomers it canbe determined which isomer is hydrolyzed. The enzymehas an almost absolute stereospecific ity for the L-D isomerwhich defines the three-dimensional arrangement of theprotein groups interacting with the substituents on the o(carbon and the catalytic groups adjacent to the P-amidewithin rather narrow limits. While the L-L isomer ofdiaminosuccinic acid monoamide is not detectably hydro-lyzed, the L-L isomer of P-methylasparagine is attacked at arate 0.2 to 0.3 % of that observed for L-asparagine, suggestingthat a P-CH3 group can be accommodated in this configura-tion while a ,&NH2 cannot.

The enzyme L-asparaginase (L-asparagine amidohydrolase,EC 3.5.1.1) has been the object of increasing interest since thepurified enzyme has been found useful in the treatment of acutelymphoblastic leukemia (l-4). Its clinical utility is attributedto the asparagine-dependent nature of these neoplastic cells incontrast to normal cells which can synthesize asparagine (4).The physicochemical properties of the crystalline enzymes fromEscherichia coli B and Erwinia caratovora have been reported onextensively (5-7) _ The protein is a tetramer of molecular Tveight-133,000 (4, 5, 7).

A variety of alternate substrates for the enzyme have been de-scribed including /3-cyanoalanine and the P-hydroxamate of L-aspartic acid both of which are converted to L-aspartic acid (4).Catalytic decomposition of 5-diazo-4-oxo-L-norvaline as well asactive site labeling by this reagent have been reported (8). This

* This work w as supported by Grants AM-09070-08 and CA-10748 from the National Institutes of Health and Grants IC-64Land PR-27 from the American Cancer Societv.

broad range o f act ivi ty has been extended to substrates contain-ing 2 asymmetrical carbon atoms which show large changes inoptical act ivity accompanying hydrolysis of the amide. Fromthe changes in the substrate Cotton ef fec t during hydrolysis ithas been possible to define the absolute stereochemical require-ments of this enzyme in positions 2 and 3 of the substrate.

MATERIALS AND METHODSXubstrates and Enzymes-L-Aspartic acid, L-asparagirre, and L-

leucine were chromatographically pure (Schwarz-Mann, NewYork). L-Leucine amide was a gif t of Professor Sofia Simmondsof Yale University. D-L, L-n-Diaminosuccinic acid monoamideand L-L, D-D-diaminosuccinic acid monoamide were synthesizedby Dr. Pauline Chang, Yale Univers ity, and the syntheses willbe reported in detail elsewhere (9). P-Methylasparagine (L-L,D-D) was also synthesized by Dr. Chang starting with commercialthree-DL-fi-methylaspartic acid (Nutritional Biochemicals, Cleve-land, 0.). Asparaginase (270 units per mg) from E. coli B wasprepared by Merck and Co. and supplied as a lyophilized sampleof an electrophoretically homogeneous preparation. One unitof asparaginase is the amount of protein required for the releaseof 1 pmole of ammonia per min from L-asparagine at pH 8.5, 37.

Optical Rotatory Dispersion and Circular Dichroismn-ORDrwas recorded with a Cary 60 recording spectropolarimeter. CDleas recorded with a Cary 61 spcctropolarimeter. Slit widthswere programmed to maintain constant light intensity. The CDinstrument was calibrated with an aqueou:: solution of d-10-camphorsulfonic acid (J. T. Baker Co.). en - Ed = 2.20 at 290nm. All measurements were carried out at 25. ORD is ex-pressed as specif ic rotation, [a], or as molecular rotation, [M] =[a]M.W./lOO, in units of dcg cm2 per dccimole. CD is espressedin terms of molecular cllipt icity, [e] = 2.303 (45OO:l7r) (EL - en)with units of dcg cm2 per decimole.

RESULTS

Substrate and Optical Properties-The primary substrate em-ployed iu this study was the monoamide of diaminosuccinic acid(Scheme I). Since there are 2 asymmetrical carbon atoms thecompound can exist as four different stereoisomers,

1 The abbreviations IIsed are: ORD, optical rotatory dispersion;CD, circular dichroism.

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1742

0 NH,H 0Ii I I II-o-c-c-c-cI I \H NH2 NH,D L

orI, D

SCHEME ILL, nn, LD, and DL. For convenience in comparing this cornpound to asparagine the carbon adjacent to the carboxyl is desig-nated as the (Y carbon and that carrying the amide as the fi car-bon. The parent compound, diaminosuccinic acid exis ts as threestercoisomcrs LL, DD, and the meso form (DL = LD). Since thefirst two isomers can be readily separated from the meso form, themeso form of diaminosuccinic acid has been used for synthesis ofthe amide substrate. The product should contain two meso-typeisomers of diaminosuccinic acid monoamide, ~-L-P-D and (Y-D-@-L(Scheme I). The product of this synthes is is not optically active(Fig. 1) confirming that the product must contain either tlvoisomers showing equal and opposite rotations, or a single isomerwith two asymmetrical centers (of opposite configuration) whichmake equal contributions to the rotation. Since the amidechromophore will not make the same contribution to rotation asthe carboxyl chromophore (see below), the firs t alternative ap-pears more likely and suggests the presence of the two isomers inequal concentrations as expected from the synthesis. Sincehydrolysis of only one of the isomers is expected to be catalyzedby the enzyme, complete enzymatic hydrolysis should be ac-compallied by the appearance of optical act ivi ty at the wavelength of the absorption bands of the unhydrolyzed amide. Asolution of the diaminosuccinic acid amide treated with asparagi-nase for 24 hours shows a large negative Cotton effec t centerednear 222 nm (Fig. 1). This suggests that only 50% o f the sub-strate is hydrolyzed as has been confirmed by determining am-monia release by Nesslerization (9). Enzymatic hydrolysis re-sults in the release o f only 50% as much ammonia nitrogen as doescomplete acid hydrolysis of the substrate (9). Once maximumrotation is achieved the CD spectrum remains the same indefi-nitely suggesting that the other isomer is completely resistant tohydrolysis.

Rate of Change of Ellipticity as Function of Enzyme Concentra-tion-Enzymatic hydrolysis of 50% of the mixture of mesoiso-mers of diaminosuccinic acid monoamide yields a product whichhas a combined molar ellip ticity at the minimum of the CDspectrum of -6.3 X lo2 deg cm2 per decimole (Fig. 1). Com-plete enzymatic hydrolysis of a 6.85 mM solution (1 mg per ml)observed over a l-cm path yields a change in observed ellipticityof -0.0445 at 222 nm. Appearance of the 222.nm Cotton eff ectis directly related to enzymatic hydrolysis of the substrate, sincethe rate of appearance of this Cotton ef fec t is directly propor-tional to enzyme concentration (Fig. 2). Time courses for theincrease in negative ellipticity at 222 nm at three different en-zyme concentrations are shown. The rate function over a 6-foldrange o f enzyme concentration is shown in the inset to Fig. 2.The pen period on the Cary 61 was set at 1 s to insure rapidpen response. The resulting large random noise does not, how-ever, reduce the precision of the slope over that of determina-tions taken at longer pen periods with less random noise. Thusthe change in cllipticity can be adapted as a satisfactory assay of

6

13

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210 220 230 240 250 260x, nmFIG. 1. ORI1 (CWW 1) and CD (Curve 2) spectra of asolution of

~-D-&I- and LY-r,-P-D-diiLmirlosI1ccinic acid monoamide (1.84 XIOP RI) after 24 hollrs incrlbation with 10 ~1 of nsparaqinase (0.164mg per ml). The WTO T bars on the axis indicate the ranges of abase-line Cl> sprctrmn taken on the same solution untreated withenzyme. Condilions: 0.05 M Tris, pH 8.0, 25, 0.2.cm path length.

enzyme activi ty. l-llfortunately in the caec of the mixture ofa-L-&D- and ol-1)-P-L-diamirlosuccinic acid monoamide the1 c issevere subst~rate inhibition at substrate concentrations >5 x1OW X. This inhibition prevented a complete kinetic analysis.The maximum turnover observed for diaminosuccinic acid mono-amide n-as 4.75 X 10 moles of substrate hydrolyzed per min permolt of enzyme in 0.05 nr Tris .HCI , pH 8.0, at a substrate concen-tration of 1 X 1OP nr. This number is the same order of nlagni-tude as the turnover lrumbcrs reported for L-asl)umgine as sub-strate under similar conditions (4, 5). The highest maximumvelocity rcportcd for t,he hydrolysis of L-asparagine is 8.95 x 104moles per min per mole o f enzyme, as assayed by the coupledspectrophotomctric assay (5). The substrate inhibition may re-late to the prcscncc of the unhydrolyzcd isomer and it is possiblethat with a pure isomer this CD assay could be devclopcd into onesatis factory for a complet~c kinetic analysis. It would have theadvantages of a direct spcctrophotometric assay without theaddition of other enzymes as in the assay coupled to the reductionof pyridine nucltotidc (1 I) or the analytical problems of mcnsur-ing ammonia release.

Stereochemistry of Isomer of Diaminosuccinic Acid MonoanzideHydrolyzed by Aspclraginase-The Cotton effect gcncrated b>50 y0 hydrolysis of the misturc of mcsoisomers of diaminosuccinicacid monoamidc is negative (Fig. I). Deduction of the sterro-chemist ry of the hydrolyzed and unhydrolyzed isomers from thesign of this Cotton cf fcct requires both an assignment of thistransition and a knomlcdge of its sign in cnvironmcnts of knownsymmetry. This can readily be deduced from the CD of somemodel compounds. Aliphatic amino acids of the L configurationiucluding r,-aspnrtic and L-glutamic acids have positive Cotton

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effect s near 200 nm due largely to the overlapping r + 7r* and n+ r* transitions of the carboql chromophore at the cy posi-tion (Fig. 3). The n + r* transitiou of the cnrbosyl group isusually near 204 nm (10). If the p- or y-carboxyl is changed toan amide chromophore as in L-asparagine there is a small but sig-nificant change in the rotation (Fig. 3). The change in clliptic-ity from the amide to the acid is relatively small, since it is due tothe vicinal eff ect of the asymmetrical center located 2 carbonatoms away. The relatively small change in optical rota tory

200 100T IME, 5ccFIG. 2. Changes in observed elliptici ty, Bobs, at 222 nm as afunction of time afler additions of 0.85, 1.76, and 2.64 units ofnsyaraginasc (0.164 mg per ml) to a solution of WI-B-D-, a-~-p-~-diaminosuccinic acid monoamide.2HBr (4.8 X 1OP M) containedin 0.05 M Tris, pH 8.0, 25. Inset, Bobs per min as a function ofasparaginasc concentration, 0.44 unit/l0 ~1. To insure adequatepen response the time constant on the Gary 61 was set at I s.

+3

2

nb I-x

G

0

-I

200 225 250X,nm

IGo. 3. CD spectra of L-asparagine (- - -), L-xspartic acid(--), and L-aspnragine after complete hydrolysis by asparaginase(.. ..) . Conditions: 0.05 M Tris, 3.75 X 10M3 M amino acid, 25,

1743strength and the low wave length of the change near 210 nm,make it unsatisfactory to follow asparagine hydrolysis wit.h op-tical rotation (Fig. 3). However, if an amide is substituted forthe cu-carboxyl, the n ---) T* transition shif ts to the region of220 nm with a resultant change in the CD pattern in the regionof 220 nm. This is illustrated in Fig. 4 by the CD spectra ofL-leucine and L-leucine amide. The free acid shows the typ i-cal positive Cotton eff ect at 198 nm, but a prominent negaOiveband appears at 227 nm in the amide. The latter can be as-signed to the n ---) 7r* transition which undergoes a pronouncedred shift in amides (10). The n --j P* Cotton eff ect is negativewhen the amide is adjacent to a carbon in the L configuration.Thus in the case of the diaminosuccinic acid monoamide hydro-lyzed by asparaginase, the Cotton eff ect at 222 nm is due t,o theunhydrolyzcd amide and that amide is of the L configuration(Fig. l), hence the L-D isomer must be the one hydrolyzed. Therelationship of this finding to the stereochemistry of the activesite of the enzyme will be discussed below (SW discussion).

If a mixture of ~-L-,&L- and a-D-fl-n-diaminosuccinic acid mon-oamide 2HBr (2.7 mg per ml) is treated with up to 0.10 mgpermlof asparaginase no optical rotatory changes are observed over aperiod of 72 hours. Furthermore, the enzyme did not release asignificant amount of ammonia from the mixture of C&L anda~-/?D isomers over this time period.

Hydro lysis of ,L-Methylasparagine-Although no hydrolysis ofthe a-~-p-~ and (Y-D-@-D mixture of diaminosuccinic acid amidewas detected, a very slow hydrolysis of the ~-L-P-L and ~-D&Disomers of fl-methylasparagine was observed. This mixture ofisomers can be prepared by amidation of the commercially avail-able three-nL&methylaspartic acid. The Cotton effect s appear-ing upon treating a mixture of (Y-L-~-L- and ac-D-fl-D-P-methyl-asparagine with asparaginase are shown in Fig. 58. A largenegative elliptic ity band appears at 204 nm and an apparentsmaller negative elipticity band at ~220 nm (Fig. 5A).

I I\ II- 0c-o-

0--- IIC-NH2

t x5227

1 I I I I I I I I I200 225 2

X,nmL50

FIG. 4. CD spectra o f L-leucine (---) and L-leucine amide(---). Conditions: 0.05 M Tris, 3.S X 10-a M amino acid, 25,0.2-cm path. 0.2-cm path.

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1744At the enzyme concentration required to observe significant

hydro lysis, holr-ever, the enzyme itse lf makes a significant contri-bution to the CD spectrum and the ellipticity at 220 nm has alarge contribution from the enzyme. Curve 1, Fig. 58, is largely

1.50

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: 0o-x

s-6

-12

I 1 I I I I I I

A

200 225X,nm

1 I I I I I I

PRODUCT ACIDL-L B -methyl aspartic acid\ \ \ \ \ \ .

UNHYDROLYZED AMIDED-D B-methyl asparagine

6I I I I I I I

i250

190 200 210 220 230 240 2501 , nm

FIG. 5. A, CD changes as a function of time during the hydroly -sis of a mixture of ~-L-P -L- and ol-u-p-D-(3-m ethylasparagin e byasparaginase. Conditions: 0.05 M Tris, pH 8.0, 1.54 X IOP M@-methylasparagine, 7.6 units (0.028 mg) if aspaiaginase per ml,O.l-cm path. Curve 1 taken immediatelv after addition of theenzym e-is largely ellipt icity contributed by the protein since atthis concentration of enzyme the ultraviolet Cotton e ffects of theprotein are detected . Curve !Z is after 110 min of incubation at 25;Curve 3 after 140 min, Curve 4 after 24 or 48 hours. The error barson the axis indicate the ranges ot a base-line CD spectrum takenon the same solution untreated with enzyme. B, ultraviolet CDof cr-L-&L&methylaspartic acid (- - -) and a-D-p-D-p-methyl-asparagine (-). A4 mixture of a-L-~-L- and a-D-p-D-p-methyl-asparagine was treated wit h aspnraginase until complete hydroly -sis had taken place (509 ammonia release). The mixture wasthen applied to a column of Dowex I-X2 to separate the productacid from the unhvdrolvzed amide. Conditions for CD as inI .Fig. 5A.

due to the enzyme. In the case of fl-methylasparagine no clearn * @ amide transition is separated out between 220 and 230 nmas in the cnsc of the diaminosuccinic arid amide (Fig. 1). Thusin order to be certain of the stereochemistry of the isomer hy-drolyzed, it was necessary to separate the product acid from theunhydrolyzed amide. This was accomplished with a Dowes I-X2 column and the CD spectra of the resolved compounds areshown in Fig. 5B. The unhydrolyzed amide is of the WD+-D con-figuration, giving rise to large negative Cotton effect s, while thehydrolyzed isomer is of the cy-L-~-L configuration, giving rise tolarge positive Cotton eff ect s. The net increase in negative ellip-tic ity observed on hydrolysis (Fig. 5A) is due to a decrease inmagnitude and slight blue shif t of the positive Cotton effect s forthe a-L-&L-acid compared to the oc-r-/-L-amide. These shi ftsin CD between the acid and the amide are more like those occur-ring on hydrolysis of asparagine (Fig. 2) where no clear separationof the 7r + QT* and n + r* transitions is present,

The ~-L-&L isomer of P-methylasparagine is hydrolyzed only0.3% as fas t as the a-L-,&r-diaminosuccinic acid monoamide asshown b y the time course for dcvclopment of the Cotton effect sin Fig. 5A. The turnover number calculated from this data isonly 150 moles of substrate hydrolyzed per min per mole of en-zyme. This is significantly faste r, however, than the hydrolysisof the ~-L-,&L isomer of diaminosuccinic acid amide.

DISCUSSIONThe bond hydrolyzed in the case of the natural substrate for

asparaginase is part of a chromophore located 2 carbon atomsaway from the asymmetrical center, and hence the rotatorychanges accompanying this hydrolysis are qualitatively less sig-nificant than if the amide bond hydrolyzed involved a carbon ad-jacent to an asymmetrical center (Figs. 3 and 4). The diamino-succinic acid derivative maintains the substrate requirements forasparaginasc, but introduces an asymmetrica l carbon adjacentto the carbon of the hydrolyzed amide bond. This greatly en-hances t,he rotatory change near 225 nm on hydrolysis as well asmoving the change to the more speci fic n ---) rr* transition of theamide (Fig. 1). Such a procedure may be applicable to the sub-strates of other enzymes where the development of assays basedon change in optical act ivi ty are desirable.

Since the natural substrate for the enzyme is L-asparagine andD-asparagine is poorly hydrolyzed (4, II), the proper relationshipbetween the enzyme-binding groups of the substrate and thecataly tic centers on the enzyme resulting in the hydrolysis of theP-amide presumably requires the L-configuration about the czcarbon. Since the p carbon is not asymmetrical the amide canassume all positions of the D or L configuration of the /3 carbon.Whether there is a strict stereospecifici ty for the P-amide oncethe substrate is in place on the enzyme surface cannot be deter-mined.

In the case of diaminosuccinic acid amide, stereospeci ficity isconferred on the fl carbon, and thus the enzyme active site mightaccept only one of the /3 carbon configurations. When the mix-ture of isomers is of the oc-u-/-L-NH2 and ~~-L-P-D-NH~ configura-tions, the c~-L-P-D-NH~ configuration is hydrolyzed (Fig. I).This is in keeping with the analogy to asparagine where the con-figuration around the carbon carrying the carboxyl must be L.The significance of the D configuration at the amide in the rapidlyhydrolyzed substrate was not apparent until enzymatic hydrolysisof t,he alternate pair o f isomers, LY-L-P-L-NH~ and o(-D-,KD-NH~was attempted. Ko significant hydrolysis was detected andthus the CGL-/!-L-NH~ isomer does not meet the requirements.Hence t,he cataly tic region o f the enzyme interacting with the

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1745

L-D. FORM

FIG. 6. Stereodrawing of the conformations of the ~-L-P-D (A)and ~-L-O-L (B) isomers of diaminosuccinic acid monoamide. Thehypothetical cataly tic groups of the enzyme are represented as aBase B and an acidic group AH. The isomers are both shown inan extended form with the --NH2 groups on opposite sides of theC-2-C-3 bond and occupying the same positions in the ~-L-P-Dand ~-L-@-L configurations. This results in the susceptible amidebond pointing away from the catalytic groups in the ~-L-@-Lisomer.amide group and the site on the protein accepting the bindinggroups around the Q: carbon arc located in the configuration shownby the a-L&n-isomer. Both the cu-carboxyl and a-amino groupshave been implicated as having binding interact ions with theprotein in the case of asparaginase from E. carotovora (12).

It appears that the L configuration of substituents at. the flcarbon interferes with the proper positioning of the substrate ifthe binding groups at the OLcarbon are confined to the L configura-Dion. If the amide group of the (Y-L-/-L isomer is to be placed inthe same position as that o f the eff ect ive ly hydrolyzed ~-L-@-Disomer, then the p-amino group occupies the position occupied bythe P-hydrogen in the (Y-L-P-D isomer. This s ite may accommo-date a hydrogen in this position but not an amino group. Xter-natively if binding requires that the substituents on the cx carbonbe in the L configuration and that the amino group of the 0 car-bon be in the position it occupies in the a-~-p-~ isomer, then theplacement of the P-amino group in this same position for the cy-L-@-L isomer results in rotation of the amide away from the site

relationships for t,he (Y-L-/-U, a-~-p-~ pair are illustrated in thestereodrawing in Fig. 6.

The ~-L-/~-II isomer is shown in the extended form, since this isthe likely form in solution with the amino groups on opposite sidesof the C-2-C-3 bond. The relationship of the binding pointson the protein receiving the groups around the cycarbon (carboxyland amino) and the catalyt,ic groups (/l and U) attacking theamide bond is then as shown by the a-~-,&n isomer in Fig. 6.This stereochemistry is not absolutely fixed in the sense that theconfiguration of the a-L-p-n isomer can change from that shownin Fig. 6 by rotation about the a-P C--C bond. Although exten-sive deviation from the extended form dots not appear like ly, theenzyme-substrate dissociation constant apprars to be relativelysmall and binding could force some changes from the extendedconfiguration . Such rotation does not change the general fea-tures of substrate-enzyme stereochemistry pictured in Fig. 6.

While the p-amino group in the C+L-P-L isomer o f diaminosuc-cinic acid amide appears to interfere with the proper positioningof the substrate, a methyl group can be accommodated in this po-sition, although the rate of hydro lysis is much decreased over therate observed if a hydrogen occupies this position. Steric factorswould appear to be primarily responsible for the decreasing rate ofhydrolysis in the order H > CHB > NH2 when these groups in thep position are placed in the L-configuration. These steric factorsmay influence the binding step or alter the relationship betweenthe catalytic groups and the substrate once the latter is bound.The cataly tic groups are represented in purely hypotheticalfashion as a base, B, attacking the carbonyl carbon, perhaps as-sisted by donation of a proton from a protein group, AH, to theamide. A specific nucleophilic attack is pictured, since data onl*O exchange (12) and on the formation of fi-aspartohydroxamicacid catalyzed by the enzyme (12, 13) have suggested but notproved the formation of an acyl enzyme intermediat,c during hy-drolysis by asparaginase.

Acknowledgments-The technical assistance of Miss JudithPascale and Miss Celeste 9. Gaumond is much appreciated. Wethank Miss Phyllis Salzman for separating the isomers of fl-methylasparagine. We are indebted to Dr. Jesse &hilling of theDepartment of Molecular Biophysics and Biochemistry for pro-gramming the stereodrawings.

REFERENCES1. KIDD, J. G. (1953) J. Exp. Med. 98, 565-6052. BROO~SE, J. D. (1961) Nature 191, 1114-11153. MASHBURK, L. T., AND WRISTON, J. C. (1964) Arch. Biochem.

Biophys. 106, 451-4524. WRISTON, J. C. (1971) Enzymes 4, 101-1215. Ho, P. P. K., MILIKIN, E. B., BOBBITT, J. L.. GRIXN.LK, E. L..BURCK, P..J., FRANK, B. fK., BOECI~, L. lb)., AND SQUIRES;R. W. (1970) J. Biol. Chem. 246, 3708-37156. FRANK, g. H. ; PEKAR, A. H., VI&OS, A. J., AND Ho, P. P. K.(1970) J. Biol. Chem. 246, 3716-37247. CAMMACK, K. A., MARLBOROUGH, D. I., AND MILLER, D. S.(1972) Biochem. J. 126, 361-3798. JACKSON, R.C. , AND HANDSCHUMACHER, R. IZ. (1970) Biochem-

istry 9, 3585-35909. HANDSCHUMACHER, R. E., GAUMOND,~. A., AKD CHANG, P. C.(1972) Biochem. kiophys. Res. Cor&zun.,.in press

10. JAFFE. H. H.. AND ORCHIN. M. (1962) Theoru and Abdications~ I