Purification and properties of certain glutamic acid-metabolizing enzymes from cockroach-muscle mitochondria

BIOCtIIMICA ET BIOPHYSICA ACTA

213

BBA 1218O

PURIFICATION AND PROPERTIES OF CERTAIN GLUTAMIC ACID-METABOLIZING ENZYMES FROM COCKROACH-MUSCLE MITOCHONDRIA" R I C H A R D R. M I L L S AND D O N A L D G. C O C H R A N

Department of Biochemistry and Nutrition and Department of Entomology, Virginia Polytechnic Institute, Blacksburg, Va. (U.S.A.) (Received O c t o b e r 2nd, i962 )

SUMMARY

Three glutamic acid-metabolizing enzymes have been identified and partially purified from cockroach thoracic muscle mitochondria. The enzymes are glutamic dehydrogenase, glutamic-aspartic transaminase and glutamic-alanine transaminase. The glutamic dehydrogenase appears to be NAD + specific, while the two transaminases require pyridoxal phosphate. Substrate specificity, pH optima, and several other characteristics of each enzyme have been determined. INTRODUCTION

The metabolism of glutamic acid by living systems is recognized to be of importance because of the pivotal position occupied b y glutamate and its versatility in entering into various types of enzymically catalyzed reactions. Perhaps the best known of these reactions is that catalyzed by the enzyme glutamic dehydrogenase in which glutamate is oxidatively deaminated to a-ketoglutaratel, 2. However, glutamate also enters into various transamination reactions whereby its amino group is transferred to a keto acid resulting in the formation of the corresponding amino acid and aketoglutarate3, 4. In addition, glutamate can be enzymically decarboxylated to produce 7-aminobutyric acid 5, or it can be aminated to produce glutamine 6. The enzymes which catalyze these reactions have been shown to occur in a wide variety of organisms including insects 1-1a. In view of the multiplicity of reactions into which glutamate m a y enter, the question arises whether more than one o f these reactions m a y occur simultaneously in a given tissue or cytological entity. In certain vertebrate tissues the answer to this question appears to be in the affirmative. For example, BOYD14 has shown that in rat liver both glutamic dehydrogenase and glutamic-aspartic transaminase are present not only in the whole homogenate, but also in each of the other commonly isolated cellular fractions. Similar work on insects has not yet been accomplished although both of these enzymes as well as glutamic-alanine transaminase have been shown to be present in the same species TM. * F r o m a t h e s i s p r e s e n t e d b y R. R. MILLS in p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s for t h e M.S. degree a t t h e Virginia P o l y t e c h n i c I n s t i t u t e , J u n e 1962.

Biochim. Biophys. Acta, 73 (1963) 213-221

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R. R. MILLS, D. G. COCHRAN

The purpose of the present investigation was to identify, separate, and characterize the glutamic acid-metabolizing enzymes present in the sarcosomes from the thoracic muscles of the American cockroach, Periplaneta americana (L.). Three principle activities were found to be present: glutamic dehydrogenase, glutamicaspartic transaminase, and glutamic-alanine transaminase. EXPERIMENTAL

Methods Preparation of mitochondria: The sarcosomes used in these experiments were prepared from isolated thoraces of adult male American cockroaches which had been adults for more than 7 days. Tile alimentary canals were carefully removed from the thoraces to avoid contamination from this source. Upon isolation, the thoraces were immediately placed in ice-cold 0.25 M sucrose. From this point the isolation proceeded by the method previously described 15. Upon completion of the isolation, the mitochondria were resuspended in 0.25 M sucrose and gently dispersed in a ground-glass, hand homogenizer. This preparation was used in the studies requiring intact mitochondria. Preparation of acetone powders: Acetone powders of the mitochondria were prepared by slowly dropping the mitochondrial suspension into a rotating erlenmeyer flask containing 50 times the suspension volume of acetone at o °. The excess acetone was filtered off, and the powder was washed and resuspended in 0.05 M phosphate buffer (pH 7-5) for use, or dried with ether for storage. Enzyme fractionation: Enzyme separation was accomplished by a sodium sulfate fractionation of the resuspended acetone powders. The glutamic-alanine transaminase was precipitated between the concentrations of o.o and 1.8o M; the glutamic acid dehydrogenase between 1.81 and 2.75 M; and the glutamic-aspartic transaminase between 2.76 and 4.50 M. Fractionation was carried out at room temperature, but filtration of the precipitate was done at o °. The resulting precipitates were immediately homogenized in 0.05 M phosphate buffer (pH 7.5) and dialysed for 4 h against 0.05 M phosphate buffer. These preparations were used in the characterization studies. Glutamic dehydrogenase assay: 3o/~moles of glutamic acid and of NAD + were incubated with various concentrations of the enzyme in 0.05 M phosphate buffer adjusted to p H 8.2. The final volume of 2.0 ml was obtained by adding o.I M phosphate buffer at pH 8.2. The reaction was followed qualitatively by paper chromatogr a p h y using the water-n-butanol-formic acid ( 1 5 : 1 o : 2 ) system. Spots were located with a 4 % solution of bromocresol purple with color being developed by ammonia. Quantitation was achieved by assaying for the formation of a-ketoglutarate at 250 m# in a Beckman DU spectrophotometer. For comparative purpose the 2,4-dinitrophenylhydrazine derivative was also prepared and assayed. The derivative was extracted in 5 ml of ethyl acetate and 3.2 ml of IO% sodium carbonate. 0.8 ml of 2.4 N N a O H was added to the carbonate fraction which was then allowed to stand for IO rain. The resulting color complex was assayed at 525 m# using a Spectronic-2o colorimeter. With both methods known samples were analyzed concurrently with each unknown. The two methods gave results which were in close agreement. The stoichiometry of the reaction was corroborated by measuring the disappearance of Biochim. Biophys. Acta, 73 (1963) 213-22x

GLUTAMATE METABOLISMBY INSECT SARCOSOMES

215

glutamate (as described later) or by the reduction of NAD + at 34 ° m#. 15 #g of bovine serum albumin were added to stabilize the NAD +. Glutamic-aspartic transaminase assay: 3o/*moles of a-ketoglutarate and of aspartic acid, 0.5/,mole of pyridoxal phosphate and i.o/,mole of MgC12 were incubated with various concentrations of enzyme in 0.o5 M phosphate buffer at p H 7.8. The final volume of 2.o ml was obtained by adding o.I M phosphate buffer at p H 7.8. The reaction was assayed for the formation of oxaloacetate at 280 m/, in a Beckman DU spectrophotometer according to the method of GREEN et al. a, Another method of analysis involved the formation of a 2,4-dinitrophenylhydrazine derivative. The derivative was extracted with 6 ml of ethyl acetate and 2.5 ml of lO% sodium carbonate. The phenylhydrazone was hydrolyzed in o.I N HC1 and analyzed at 280 m/, in a Beckman-DU spectrophotometer. Known samples were analyzed concurrently with each unknown for comparison. Glutamic-alanine transaminase assay: The reaction mixture for this assay was the same as for the glutamic-aspartic transaminase reaction except that alanine was substituted for aspartic acid and the p H was 7.6. The formation of pyruvic acid was used to follow the course of the reaction. Pyruvate was determined by the 2 % salicylaldehyde method of GREEN et al. 4. A m i n o acid analysis: Analysis of amino acids was accomplished by both oneand two-dimensional descending chromatography on W h a t m a n No. I paper. T h e two-phase system of distilled water-n-butanol-formic acid (15:1o:2) was used in all analyses. The second solvent system for the two-dimensional work consisted of the one-phase system distilled w a t e r - N H a O H (15:1). Quantitation of the amino acids was accomplished b y the cadmium chloride method of MORTREUIL AND KHOUVINE ls. Protein determination: Protein concentration of the various enzyme preparations was determined b y the method of LOWRY et al. 17. Crystalline bovine serum albumin was used as the standard protein. Materials

The leucine, valine, isoleucine and a-aminobutyric acid were products of Nutritional Biochemicals Company; crystalline bovine serum albumin was obtained from Pentex, Inc. ; all other biochemicals were purchased from California Corporation for Biochemical Research. Common laboratory chemicals were of reagent grade. Demineralized distilled water was used in the preparation of all solutions.

RESULTS Glutamate metabolism with intact mitochondria

In previous work it has been shown that intact sarcosomes from the American cockroach are capable of oxidizing glutamic acid at a rapid rate TM. Coupled with this oxidation is the esterification of phosphate into high-energy phosphate bonds. F r o m this work it was assumed that glutamic dehydrogenase is present in these muscle mitochondria as it is in most other mitochondria 14. Upon closer examination of the reaction products, however, it was discovered that the principle product is aspartic Biochim. Biophys. Acta, 73 (1963) 213-221

210

R. R. M I L L S , D. G. COCHRAN

acid. This finding clearly indicated the possibility of a transaminase enzyme in the sarcosomes. To explore this possibility further a series of experiments was undertaken in which several of the Krebs-cycle intermediates between a-ketoglutarate and oxaloacetate were incubated with intact mitochondria both in the presence and in the absence of glutamate. The results are shown in Table I. It is evident from these TABLEI FORMATION OF OXALOACETATE OR ASPARTATE BY INTACT MITOCHONDRIA E a c h r e a c t i o n vessel c o n t a i n e d 3° / z m o l e s of e a c h s u b s t r a t e i n d i c a t e d b e l o w as well as MgCI,, 2o#moles; phosphate buffer (pH7.4), 4o/*moles; ADP, Io/zmoles; mitochondria (2-3mg p r o t e i n ) ; h e x o k i n a s e , 15o K M u n i t s ; g l u c o s e , 5 o / z m o l e s ; s u c r o s e , 0.25 M t o a f i n a l v o l u m e o f 2.3 m l . T e m p e r a t u r e , 2 5 ° ; t i m e , 3 ° m i n .

Substrate

Glutamate a- K e t o g l u t a r a t e a-Ketoglutarate plus glutamate Succinate Succinate plus glutamate Fumarate Fumarate plus glutamate Malate Malate plus glutamate

Oxaloacetate formed ( tzmoles)

A spartate formed (l,moles)

-7 -8 -8 -12

7 -5 -5 -5 -IO

findings that a transaminase reaction does occur in these mitochondria, and that glutamate is an essential reactant. The data also give direct evidence that each step in the Krebs cycle between a-ketoglutarate and oxaloacetate is present and functional in these mitochondria. This work formed the basis for the purification and characterization studies which follow.

Purification The three principle enzyme activities found in the acetone powders of the cockTABLE

II

ENZYME-PURIFICATION CHART SHOWING SPECIFIC ACTIVITY, YIELD, AND PURIFICATION

Enzyme

Glutamic dehydrogenase acetone powders Glutamic dehydrogenase from fractionation Glutamic-aspartic transaminase acetone powders Glutamic-aspartic transaminase from fractionation Glutamic-alanine transaminase acetone powders Glutamic-alanine transaminase from fractionation

Product formed (#moles]h)

Protein (rag)

Specific activity

Yield (%)

Fold purification

45 .o 36.0

37 12

1.2 3.0

-80.0

-2. 5

3°.o

37

0.8

--

--

24.0

io

2.4

8o.0

3.0

7.o

37

o.2

--

--

5.5

8

o.7

68.8

3.6

B i o c h i m . B i o p h y s . A c t a , 73 (1963) 2 1 3 - 2 2 1

GLUTAMATE METABOLISM BY INSECT SARCOSOMES

217

r o a c h m i t o c h o n d r i a are g l u t a m i c d e h y d r o g e n a s e , g l u t a m i c - a s p a r t i c t r a n s a m i n a s e , a n d g l u t a m i c - a l a n i n e t r a n s a m i n a s e . These activities were s e p a r a t e d b y NazSO 4 f r a c t i o n a t i o n and, as e x p e c t e d , in the process some purification of t h e activities was achieved. T h e specific a c t i v i t y , p e r cent yield, a n d fold purification are o u t l i n e d in T a b l e I I . All of t h e s u b s e q u e n t d a t a were o b t a i n e d using these p a r t i a l l y purified enzyme preparations.

Glutamic dehydrogenase T h e g l u t a m i c d e h y d r o g e n a s e - c a t a l y z e d r e a c t i o n was s t u d i e d in t h e f o r w a r d d i r e c t i o n b y a s s a y i n g for t h e f o r m a t i o n of a - k e t o g l u t a r a t e . I t was found t h a t t h e r e a c t i o n r a t e is linear for a p p r o x . 20 min. D u r i n g this p e r i o d of t i m e 16.o/~moles of p r o d u c t were formed. T h e e n z y m e c o n c e n t r a t i o n was 8 m g of p r o t e i n per m l of r e a c t i o n m i x t u r e . T h e r e a c t i o n was also s t u d i e d b y v a r y i n g t h e s u b s t r a t e concent r a t i o n over a wide range of values. I n this w a y d a t a were o b t a i n e d for the d e v e l o p m e n t of a LINEWEAVER-BURK plot 19. F r o m this plot the Michealis c o n s t a n t was shown to be I . I . I O -a M. The r e a c t i o n was found to be N A D + specific, N A D P + y i e l d i n g o n l y traces of a - k e t o g l u t a r a t e . T h e p H o p t i m u m for this r e a c t i o n was 8.2 (Fig. I). I t was o b t a i n e d b y i n c u b a t i n g

f

/ x× \

161'13

!

o 12

"6

\\\

8

o/

.

NO

:3,

i

7.0

i 7,4

I 7.8

i 8.2

i 8.6

i 9.0

i 9.4

pH

Fig. I. Effect of pH on reaction rate. Each of the reactions was examined using o. i M phosphate buffer adjusted to the appropriate pH values. × - - x, glutamic dehydrogenase ; C)-- ©, glutamicaspartic transaminase; O--Q, glutamic-alanine transaminase. t h e r e a c t i o n m i x t u r e in p h o s p h a t e buffer a d j u s t e d to t h e various required p H values. No a t t e m p t was m a d e to s t u d y t h e effect of different buffers on t h e p H o p t i m u m . The s u b s t r a t e specificity of the e n z y m e was e x a m i n e d b y i n c u b a t i n g p r o s p e c t i v e s u b s t r a t e s w i t h t h e e n z y m e for 60 min. The a m o u n t of a - k e t o acid f o r m e d from each s u b s t r a t e served as a m e a s u r e of the reaction. I t was shown t h a t the e n z y m e is a l m o s t c o m p l e t e l y specific for L-glutamate. L - a - A m i n o b u t y r a t e , L-leucine, a n d Lm a l a t e were a c t e d u p o n only v e r y w e a k l y , while D-glutamate, L-lactate a n d s e v e r a l o t h e r c o m p o u n d s gave no reaction. I n h i b i t i o n studies were carried o u t b y i n c u b a t i o n of c a n d i d a t e i n h i b i t o r s w i t h t h e control r e a c t i o n m i x t u r e . The inhibition was c a l c u l a t e d for each i n h i b i t o r as a p e r cent of t h e control. T h e results are s u m m a r i z e d in T a b l e I I I .

Biochim. Biophys. Acta, 73 (1963) 213-221

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R.R.

MILLS,

D . G. C O C H R A N

TABLE INHIBITION

OF

THE

GLUTAMIC

Inhibitor

None D-Glutamate Adipate Glutarate Succinate Fumarate Malate L-Aspartate n-Aspartate Hydroxylamine 2,4-Dinitrophenylhydrazine a-Ketoglutarate

III

DEHYDROGENASE

REACTION

BY

VARIOUS

INHIBITORS

Inhibitor concentration

a-Ketoglutarate formed

Inhibition

(M)

(~moles]h)

(%)

-0.3 • lO -2 o . 3 . i o -2 0.3 " 1°-2 0. 3 • l O -2 o.3" IO-2 0. 3 • l O -2 o. 3 " I o - 2 o.3 " io-2 o. 3 ' I O - 3 0. 5 - lO - 3 0. 3 • l O - 2

20 9 14 7 17 17 17 16 18 12 2 12

-55 3° 65 15 15 15 2o Io 4° 90 4°

Glutamic-aspartic transaminase Characterization of the glutamic-aspartic transaminase was conducted by incubating the enzyme with a-ketoglutarate and aspartate, and assaying for the formation of oxaloacetate. This reaction was found to be linear for approx. 18 min. I n these studies the enzyme concentration was 5-6 mg of protein per ml of reaction mixture, and approx. 12/zmoles of oxaloacetate were produced in 18 min. Dialyzed enzyme exhibited a requirement for pyridoxal phosphate. The p H optimum for the reaction was 7.8 (Fig. i), but the curve had a rather broad peak. I t has been pointed out that Michaelis constants for transaminase-type reactions, in which two substrates are involved, are not necessarily comparable to those from one-substrate reactions~°, ~1. Nevertheless, values obtained under a standard set of conditions m a y still be useful in comparative biochemistry. Thus, in obtaining data for a Lineweaver-Burk plot, the substrates were equimolar in concentration for

TABLE

IV

SUBSTRATE S P E C I F I C I T Y OF GLUTAMIC--ASPARTIC TRANSAMINASt~ 3 °/*moles

of each amino acid and 30/~moles of a-ketoglutarate were used as substrates. time was 6o rain. Protein concentration was 2 mg/ml of reaction mixture.

Substrate

Aspartate Alanine Leucine Isoleucine Valine Methionine 7-Aminobutyrate a-Aminobutyrate

Keto acid

Glutamate

Glutamic-aspartic

formed (Itmoles)

formed (~moles)

transaminase ( %)

12.O

12.O

Reaction

IOO

1.2

1.3

I .O

I .O

IO 8

--

O

O

--

O

O

--

O

- -

1. 3

IO

- -

1.3

IO

O

Biochim. B i o p h y s . Acta, 73 (1963) 213-221

GLUTAMATE METABOLISM BY INSECT SARCOSOMES

219

each point. Under these conditions the apparent Km value was 1. 5.1o -3 M. All other reaction conditions were held constant. Substrate-specificity studies were conducted by assaying for the formation of glutamate from a-ketoglutarate and the candidate amine-group donors. The results are summarized in Table IV. From the data it is evident that of all the compounds examined only aspartate gives a strong transamination. It is particularly significant that alanine does not serve as a good amine-group donor for this reaction. A group of common enzyme inhibitors has been examined for their effect on glutamic-aspartic transaminase. The studies were conducted by assaying for the formation of oxaloacetate in the presence and in the absence of candidate inhibitors. The results, shown in Table V, are expressed in terms of inhibition of the oxaloacetate formed in the control. TABLE V INHIBITION

OF G L U T A M I C - - A S P A R T I C A N D G L U T A M I C - - A L A N I N E T R A N S A M I N A S E S BY VARIOUS INI-IIBITORS

Glutamic-aspartie transaminase Glutamic-a!anine transaminase Inhibitor

None Hydroxylamine a-Ketoglutarate oxime Pyruvic oxime Oxaloacetic oxime NaAsO 2 NaF Na2SO,

Inhibitor concentration (M)

m

0. 3 • lO-2 0. 3

Product formed in 6o min ( ktmoles )

Inhibition ( %)

Product formed in 60 min (#moles)

Inhibition ( %)

I2.O

O

8.0

O

2.4

8o

1.6

80

lO -2

lO.8

IO

7.0

12

0. 3 lO -2

4.8

60

2.6

65

6.o 3.6

5° 7°

5.0 4.0

4° 5°

0. 3

10 -2

0. 3 O. 3

IO - a 10 -8

O

0. 3

10 -2

11.4

IOO

5

O

8.0

IOO

o

Glutamic-alanine transaminase In studies on this enzyme a-ketoglutarate and alanine served as the substrates. The reaction was followed by assaying for the formation of pyruvate. The reaction rate was linear for more than 60 min but the rate of product formation was quite slow. In 60 min approx. 8/zmoles of pyruvate were produced. The enzyme concentration was 5 mg of protein per ml of reaction mixture. This enzyme also has a requirement for pyridoxal phosphate after dialysis. The pH optimum of the reaction in o.I M phosphate buffer was 7.6 (Fig. I), but again the peak of the curve was rather broad. The apparent K m value for the reaction was 8.0. lO -4 M. Equimolar concentrations of the two substrates were used for each point on the LineweaverBurk plot. The enzyme is almost completely specific for alanine as the amino acid substrate. Among the other amino acids examined only serine and threonine produced detectable amounts of glutamate. Aspartic acid gave no reaction. Results of the inhibition studies are shown in Table V. A comparison of the data with those for the aspartic transaminase show some striking similarities. The per cent inhibition of the two reactions by the same inhibitors agrees very closely with the exception of NaAsO2. Even here there is only a 20% difference in the two reactions. Biochim. Biophys. Acta, 73 (1963) 2 1 3 - 2 2 I

220

R. R. MILLS, D. G. COCHRAN

DISCUSSION The results presented in this paper show that at least three glutamate-metabolizing enzymes are present in intact sarcosomes from the thoracic muscles of the American cockroach. Thus, the possibility that these several types of reactions can occur simultaneously in the intact muscle is now clearly demonstrated. It is not yet clear, however, under what conditions each reaction is important or what normally controls the rate of each reaction, although several mechanisms could be postulated. For example, the oxidation state of the nicotinamide nucleotide could determine the rate of reaction of glutamic dehydrogenase. Alternatively, the rate of removal of oxaloacetate or of pyruvate by the Krebs cycle would obviously influence the rate of the transaminase reactions. The rate at which the Krebs cycle functions m a y in turn be a reflection of the effective concentration of ADP. Regardless of the control mechanism in the intact organism, however, the finding of a complex of glutamateinvolved reactions in the sarcosomes emphasizes previous admonitions that care must be exercised in interpreting the experimental results obtained from mitochondrial-catalyzed systems, such as oxidative phosphorylation, in which glutamate is used as the primary exogenous substrate 15,22,23. In general, the partially purified sarcosomal enzymes described in this paper appear to be similar to the corresponding enzymes reported from other animal tissues, with some minor exceptions. The glutamic dehydrogenase presents a good example of this because it has several characteristics which are nearly identical with those of crystalline glutamic dehydrogenases from chicken liver 24 and from beef liver ~5. On the contrary, these three enzymes appear to differ significantly from the glutamic dehydrogenase from cockroach fat body 12 at least with respect to Michaelis constants. Thus, even in a given organism the same enzymic activity from different tissues m a y exhibit variations which quite probably are physiologically significant. The transaminases examined in this study illustrate an interesting parallel. They appear to represent two enzymic activities which are very much alike in p H optima, Michaelis constants, cofactor requirements and inhibitor relationships. Among the characteristics examined, they differ only in substrate specificity. In this regard, however, one is clearly a glutamic-aspartic transaminase and the other a glutamic-alanine transaminase. ACKNOWLEDGEMENT

This investigation was partially supported by the U.S. Public Health Service through a grant (No. E-2o67) from the National Institute of Allergy and Infectious Diseases.

REFERENCES 1 M. DAMODARAN AND I~. R. ~AIR, Biochem. J., 32 (1938) lO64. 2 j . G. DEWAN, Biochem. J., 32 (1938) 1378. 3 A. E. BRAUNSTEIN AND M. G. KRITSMAN, Enzymologia, 2 (1937) 129. 4 D. E. GREEN, L. F. LELOIR AND V. NOClTO, J. Biol. Chem., 161 (1945) 559. 5 E. ROBERTS AND S. FRANKEL, J . Biol. Chem., 187 (195o) 55. e A. MEISTER, Physiol. Rev., 36 (1956) lO 3. 7 H. J. STRECKER, Arch. Biochem. Biophys., 32 (1951) 448. s W. A. BULEN, Arch. Biochem. Biophys., 62 (1956) 173. H. J. EICHEL AND J. BUKOVSKY, Nature, 191 (196I) 243.

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10 S. P. BESSMAN, J. ROSSON AND E. C. LAYNE, J. Biol. Chem., 2 O l ( 1 9 5 3 ) 3 8 5 . lx H. BEEVERS, Biochem. J., 48 (1951) 132. 12 j . w . MCALLAN AND W. CHEFURKA Comp. Biochem. Physiol., 3 (1961) I. 18 B. A. KILBY AND E. NEVILLE, J. Biol. Chem., 34 (1957) 276. li j . W. BOYD, Biochem. J., 81 (1961) 43415 D. G. COCHRAN, Intern. Congr. Entomol. Proc. rzth, I (1961) 663. 16 M. MORTREUIL AND Y. KHOUVlNE, Bull. Soc. Chim. Biol., 36 (1954) 425 . 17 O. H. LOWRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 18 D. G. COCHEAN AND K. W. KINa, Biochim. Biophys. Acta, 37 (196o) 562. 19 H. LINP.W~.AVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. s0 A. NISONOFF AND F. W. BARNES, Jr., J. Biol. Chem., 199 (I959) 713 • st F. S. CooK, Can. J. Biochem. Physiol., 35 (1957) 1289. 22 p. BOEST AND E. C. SLATER, Nature, 185 (I96O) 5372s p. BoEsx, Biochim. Biophys. Acta, 57 (I962) 256. 24 j . E. SMOKE, J. Biol. Chem., 223 (I956) 27 I. ss j . A. OLSON AND C. B. ANFINSEN, J. Biol. Chem., 2o2 (1953) 841.

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