Characterization of porcine malate dehydrogenase I. An active center peptide

Characterization of porcine malate dehydrogenase I. An active center peptide

489 BIOCHIMICA ET BIOPHYSICA ACTA BBA 35913 C H A R A C T E R I Z A T I O N OF P O R C I N E MALATE D E H Y D R O G E N A S E I. AN ACTIVE C E N T E...

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489

BIOCHIMICA ET BIOPHYSICA ACTA

BBA 35913 C H A R A C T E R I Z A T I O N OF P O R C I N E MALATE D E H Y D R O G E N A S E I. AN ACTIVE C E N T E R P E P T I D E

E. M. GREGORY, M. S. ROHRBACH AND J. H. HARRISON

Department of Chemistry, University of North Carolina, Chapel Hill, N.C. 275I4 (U.S.A.) (Received May 24th, 1971)

SUMMARY

Iodoacetamide has been found to be a highly useful reagent for the selective chemical modification of active center residues of porcine heart mitochondrial malate dehydrogenase (L-malate:NAD÷ oxidoreductase, EC 1.1.1.37). The addition of the coenzyme, NADH, fully protects the enzyme from alkylation thus suggesting that chemical modification is occurring at or near the NADH binding site. Alkylation by this reagent leads to a totally inactive enzyme. The identification of histidine as the site of chemical modification confirms work recently reported by B. H. ANDERTON (Eur. J. Biochem., 15 (197o) 562). However, in addition to the alkylated histidine, we have isolated a small active center peptide containing 2 residues of glycine in addition to carboxymethyl histidine. The composition and structure of this peptide was identified after difficulties arose in attempts to identify the active center residues derived from proteolytic digestion of the iodoacetamide modified enzyme.

INTRODUCTION

Selective chemical modification, when utilized properly, can be an important technique in the elucidation of the nature of the active center of an enzyme. The localization of the residues to an area "at or near the active center" has generally been gained inferentially through the observed protection of the enzyme by the presence of coenzyme or substrate. This at best restricts the area of interaction to the locale of the binding sites of these molecules but at worst should be still in the area of catalysis. In order to determine the usefulness of this approach towards a study of the mitochondrial malate dehydrogenase (L-malate:NAD + oxidoreductase, EC 1.1.1.37) of porcine heart a group of reagents commonly used in specific chemical modification reactions were investigated. We have previously reported in a preliminary communication 2 the observation that iodoacetamide can be utilized to selectively carboxamidomethylate a limited number of residues (two) leading to complete inactivation Abbreviations: CAM, carboxamidomethyl; DTNB, 5,5'-dithiobis (2-nitrobenzoic acid); t-Boc, tert.-butoxycarbony].

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E.M. GREGORYet al.

of mitochondrial malate dehydrogenase. We have undertaken a study to determine the nature of this active center residue as a first step in the elucidation of the nature of the active center of this enzyme. MATERIALS

NADH, L-malic acid and oxaloacetic acid were purchased from Sigma. Iodoacetamide (Eastman, 91o8) was used without further purification. Iodo[14Cjacetamide (Amersham-Searle) containing 5o #C/mmole was used in the labelling experiments. L-Methionine (Mann and Pierce)tert.-butoxycarbonylhistidine (t-Boc-histidine) (Pierce), cysteine. HC1, and histidine (Nutritional Biochemicals) were used to prepare chromatographic standards. Cellulose thin-layer plates were obtained from Eastman Chemicals. Pronase and carboxypeptidases A and B were purchased from Calbiochem and Sigma, respectively. Aquasol, an aqeuous soluble scintillation fluid, was purchased from New England Nuclear. Dansyl chloride and dansyl derivatives were purchased from Sigma. Polyamide plates were purchased from Gallard-Schlessinger. METHODS

Enzymatic assay Porcine heart mitochondrial malate dehydrogenase was purified from acetone powders of fresh pig hearts 15. The resultant preparation exhibited a specific activity of 16oo units/ml per A280nm. The standard assay consisted of 9 ° mM sodium pyrophosphate buffer (pH IO.6), 1. 5 mM NAD ÷, 33 mM sodium L-malate; tile enzyme was added at zero time, and the absorbance increase at 340 nm was measured. Units of enzymatic activity are expressed as units of enzyme per ml, divided by the "A28o n m value", the absorption of the solution at 280 nm according to previous methods 3. Protein concentrations were determined spectrophotometrically at 280 nm utilizing a molar extinction coefficient of 17 800.

Chemical modification Malate dehydrogenase (8-3o/zmoles) was incubated with a 2oo-fold molar excess of iodoacetamide in 5 mM sodium phosphate buffer (pH 7.5) and stored in the dark to prevent light-initiated decomposition of tile reagent. The inactivation was followed by assaying for dehydrogenase activity as a function of time using the previously described standard assay system. A control sample of enzyme, incubated under identical conditions, but without any alkylating reagent, was measured for activity at each time the incubation sample was assayed. Each inhibition was followed until inhibition sample velocity was lO% of control velocity.

Proteolytie digestion of 14C-labeled malate dehydrogenase Samples of inactivated 14C-labeled malate dehydrogenase were digested enzymatieally at 4 °° for 48 h by the addition of pronase in a IO:I weight ratio of malate Biochim. Biophys. Acta, 243 (1971) 489-497

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dehydrogenase to pronase followed by the addition of a weight ratio of 2o:1 each of carboxypeptidase A and B for an additional 18 h (ref. 4). A small crystal of thymol was added to the degradation mixture to prevent bacterial growth. After digestion, the mixture was desalted by chromatography on a 0. 9 cm × 15 cm column of Dowex 5o-X8, hydrogen form, followed by chromatography on Sephadex G-io. The ninhydrin positive fractions were pooled, stripped, and stored frozen.

Preparation of carboxamidomethyl (CAM) derivatives The CAM derivatives of methionine, histidine, and cysteine were prepared by the method of GUNDLACH et al) with the following modifications. The CAM derivatives were prepared in each case by dissolving 50 mg of L-cysteine- HC1, L-methionine, or t-Boc-histidine and 15o mg of 2-iodoE14C]acetamide (15 #C) in io ml of 0.o 5 M sodium phosphate and adjusting the p H to 6.0. The solutions were protected from light and incubated at 4 °° for 24 h. In each case, excess salt and iodoacetamide were removed by desalting on a column of Dowex 5o-X8 according to the method of ANFINSEN et al. ~. The CAM derivatives in each case were eluted from the column with a 7% solution of ammonia. In the case of the t-Boc-histidine derivative, the t-Boc group was removed by the addition of BFa- diethyl ether in glacial acetic acid (2 ml in 20 ml) followed by stirring at 25 ° for I h. The deblocked histidine derivative was then desalted on Dowex 5o-X8 as above.

Thin-layer chromatography Thin-layer cellulose sheets (Eastman) were utilized without further preparation. Chromatographic solutions of chloroform-methanol-ammonia (2:2 :I, by vol.), and butanol-pyridine-acetic acid-water (68:4o:12:25, by vol.) were prepared immediately prior to usage. After chromatography, the plates were dried and developed with ninhydrin spray. The spots of ninhydrin color were excised and placed in scintillation fluid for monitoring of radioactivity.

Preparation of CAM-malate dehydrogenase peptide 25 mg of malate dehydrogenase were inactivated with iodoE14C]acetamide and proteolytically digested as described above. The digest was then lyophylysed and applied to the I5-cm column of a Beckman 116 amino acid analyzer and eluted with the standard p H 5.28 buffer. The effluent stream was diverted to a fraction collector and fractions were monitored for radioactivity, pooled, and desalted on Dowex 5o-X8.

Sequential analysis of active center peptide The sequence of amino acids in the labeled peptide isolated after proteolytic digestion of malate dehydrogenase was derived using the technique of sequential degradation plus dansylation as described by GRAY7. Dansyl derivatives were identified after separation using two solvent systems on polyamide layer chromatography as described by WANG8. RESULTS AND DISCUSSION

The effects of iodoacetic acid, iodoacetamide, N-ethylmaleimide, and 5,5'Bioch,m. Biophys. Mcla, 243 (197i) 489 497

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TABLE I SECOND

ORDER

INHIBITION

RATE

CONSTANTS

Second order rate c o n s t a n t s calculated from the inhibition of 3/~moles of malate dehydrogenase, 5 ° mM s o d i u m p h o s p h a t e (pH 7.5) with iodoacetate, D T N B , N-ethylmaleimide and iodoacetamide at 25 °.

Inhibitor

Rate constant

[odoacetate DTNB N-Ethylmaleimide Iodoacetamide

o, 22 0.24 o.44 3.5 °

dithiobis(2-nitrobenzoic acid) (DTNB) on the e n z y m a t i c a c t i v i t y of m a l a t e d e h y d r o genase have been r e p o r t e d previously 2. As can be seen in Table I, the second order r a t e c o n s t a n t s for iodoacetate, N - e t h y l m a l e i n f i d e a n d D T N B v a r y only slightly while i o d o a c e t a m i d e yields a r a t e c o n s t a n t some 8 - I 2 - f o l d greater. In view of t h e o b s e r v a t i o n t h a t i o d o a c e t a m i d e at a r e l a t i v e l y low m o l a r excess (200) a n d within 2 h t o t a l l y i n a c t i v a t e d m a l a t e dehydrogenase, this i n h i b i t o r was chosen for further studies. I n order to d e t e r m i n e the general location of i n t e r a c t i o n of i o d o a c e t a m i d e with m a l a t e dehydrogenase, inhibition studies were carried out in t h e presence of coenzyme, N A D H , a n d substrates, m a l a t e a n d o x a l o a c e t a t e . The a d d i t i o n of 0.20 mM r e d u c e d coenzyme, an N A D H to e n z y m e ratio of 13 : I, to the i n a c t i v a t i o n m i x t u r e (200 m o l a r excess iodoacetamide) y i e l d e d an e n z y m e t o t a l l y p r o t e c t e d from i n a c t i v a t i o n . N A D H t h u s a p p e a r s to r e n d e r specific residues at or near the coenzyme b i n d i n g center, a n d thus, at or n e a r the e n z y m a t i c active center, inaccessible for chemical modification b y i o d o a c e t a m i d e . I n a similar manner, t h e s u b s t r a t e s o x a l a c e t a t e a n d L-malate, while not fully p r o t e c t i n g the enzyme, reduced the r a t e of i n a c t i v a t i o n when a 2oo-fold m o l a r excess of i o d o a c e t a m i d e was utilized. A d d i t i o n of 22 mM L-malate to the i n c u b a t i o n m i x t u r e reduces t h e r a t e of inhibition a n d a second order r a t e c o n s t a n t in the presence of L-malate of I . I - I O 1 1.mole -1.rain -1 is observed. Similarly, the a d d i t i o n of 22 mM o x a l a c e t a t e was f o u n d to reduce the r a t e of inhibition a n d a second order rate c o n s t a n t of 2.7-1o -1 1.mole 1.rain-1 was d e t e r m i n e d . To q u a n t i t a t e the i n c o r p o r a t i o n of i o d o a c e t a m i d e into m a l a t e dehydrogenase, a 2oo-fold molar excess of iodo[I-i4C]acetamide was i n c u b a t e d with 3" IO ~ M m a l a t e d e h y d r o g e n a s e a n d the reaction quenched b y t h e a d d i t i o n of IOO #1 of 14 M fl-merc a p t o e t h a n o l when the a c t i v i t y of the i n c u b a t i o n m i x t u r e was 5 - 1 o % t h a t of the control. A similar e x p e r i m e n t was p e r f o r m e d in which N A D H (0.2 raM) was a d d e d to t h e i n a c t i v a t i o n m i x t u r e . B o t h t h e quenched N A D H p r o t e c t e d samples a n d the u n p r o t e c t e d samples were d i a l y z e d against 3 changes of 4 1 each of 50 mM s o d i u m p h o s p h a t e buffer (pH 7.0). The protein c o n c e n t r a t i o n of each of the s a m p l e s was then d e t e r m i n e d a n d a k n o w n fractional aliquot of t h e s a m p l e was p l a c e d in 12-15 ml of aquasol scintillation cocktail, a n d m o n i t o r e d on a N u c l e a r Chicago Scintillation counter. A b l a n k s a m p l e of an equal volume of the final dialysis buffer a n d an e x t e r n a l ~4C source were c o u n t e d w i t h each b a t c h of samples. Counting times were IO rain with 5 repetitions, a n d t h e average of t h e 5 assays, corrected for quench, was used to Biochim. Biophys. Acta, 243 (1971) 489-497

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MITOCHONDRIAL MALATE DEHYDROGENASE

calculate moles 14C bound per mole of enzyme. The unprotected sample of malate dehydrogenase incorporated an average of 2.2 moles of 14C per 7 ° ooo molecular weight based on 8 determinations. On the other hand, the N A D H protected malate dehydrogenase sample incorporated only 0.3 mole 14C per 7 ° ooo molecular weight, based on 4 determinations. P F L E I D E R E R 9 and KAPLAN1° have previously reported evidence for 2 catalytically active centers in porcine mitochondrial malate dehydrogenase per 7 ° ooo molecular weight based upon the binding of 2 moles of N A D H per mole of malate dehydrogenase. I t appears, therefore, that under the conditions employed, iodoacetamide selectively carboxamidomethylates a limited number of residues (two) at or near the enzymatic active center, an average of i residue per active center, leading to complete inactivation of mitochondrial malate dehydrogenase. The possible reactive residues in a protein which might react with an alkylating reagent such as iodoacetamide include cysteine, histidine, lysine, and possibly methionine. PFLEIDERERn has recently succeeded in labeling 4 active center residues in the cytoplasmic form of malate dehydrogenase utilizing iodoacetie acid. The residues modified have been identified by PrLEIDERER as methionine. The reaction of iodoacetamide with methionine is such that the product formed is the CAM sulfonium salt of methionine. This product is highly unstable to acid hydrolysis and yields under the conditions necessary for hydrolysis of proteins: homoserine, homoserine lactone, methionine, and carboxymethylhomocysteine. In view of the recent findings of P F L E I D E R E R , it was felt necessary to consider the possibility that methionyl residues were also modified in the mitochondrial form of malate dehydrogenase. Thus, an enzymatic method of digestion rather than acid hydrolysis was utilized to assure that destruction of methionine derivatives, if present, be minimized. Samples of malate dehydrogenase were inactivated with iodo[14C]acetamide and subjected to proteolytic digestion by pronase and carboxypeptidase A and B (see details in METHODS). Thin-layer chromatography of the proteolytically digested malate dehydrogenase was performed using two different chromatography systems. The RF values of T A B L E II

RF VALUES OF RADIOACTIVITY R F v a l u e s of t h e r a d i o a c t i v e s p o t s 14C-labeled CAM d e r i v a t i v e s of L-histidine, L-methionine, Lc y s t e i n e a n d t h e 14C-labeled i n a c t i v a t e d m a l a t e d e h y d r o g e n a s e digest, p r e p a r e d as a b o v e . I n cases w h e r e m u l t i p l e r a d i o a c t i v e s p o t s were observed, t h e figures in p a r e n t h e s e s indicate p e r c e n t a g e o f t o t a l c o u n t s in t h e c o m p o n e n t . Solvent system

[14C]CA M-

[14C]CA Mcysteine

[14C]CA Mmethionine

[14C]-labeled malate dehydrogenase digest

0.55 (56%) 0.66 (44%)

0.45

o.18 (80%)

o.18

o.ii

o.io

0.04

0.o 4

histidine

Chloroform-methanola m m o n i a (2:2 :i, b y vol.) Butanol-pyridine-acetic a c i d - w a t e r (68:4o:12:25, b y vol.)

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E.M. GREGORY et al.

the radioactive spots of the ~4C-labeled standards of cysteine, histidine, methionine, and the malate dehydrogenase digest are shown in Table II. In a solvent system of chloroform-methanol-ammonia the single radioactive spot from the digest with a RF value of o.18 is identical to the RF of standard CAM-methionine. Using a butanolpyridine-acetic acid water solvent system the RF value of 0.04 for the digest radioactive spot again is identical to the RF of the standard CAM-methionine.

1o 5 i

i

c~15 0

5 ._c E "--15

u

J

i

1:5 5 A

0

100

200 Effluent (ml)

300

4OO

Fig. I. Elution p a t t e r n s for (A) [14C]carboxamidomethylmethionine sulfonium salt, (C) 14C-labeled inhibited malate dehydrogenase digest and (B) [14C]carboxamidomethylhistidine. The eluent s t r e a m was diverted from a Phoenix one-column analyzer to a fraction collector. Fractions of 1. 5 ml were collected and counted for radioactivity. All radioactivity values were corrected for quench.

Identification of the labeled residue was also attempted by chromatography of [14ClCAM-methionine, E14CJCAM-histidine, and the digested E14ClCAM-inalate dehydrogenase on the Phoenix one-column amino acid analyzer. The effluent stream of the analyzer was diverted to a fraction collector and the fractions analyzed for radioactivity. Fig. IA represents the elution pattern for CAM-methionine. The two optical isomers eluted as a double peak at 412 and 420 m]. In Fig. IC the elution pattern for the proteolytically digested CAM-malate dehydrogenase is shown. A single symmetrical peak containing 80% of the applied counts eluted at 404 ml. Fig. I B represents the elution pattern for CAM-histidine. The peak at 164 ml probably represents 1,3-diearboxamidomethylhistidine while the two peaks at 364 ml and 372 ml represent I-N- and 3-N-carboxamidomethylhistidine. The elution position of the labeled residue in malate dehydrogenase appeared to coincide, within experimental limits, with the elution position for one of the two isomers of CAMmethionine. This observation along with the above thin-layer chromatography data originally led us to assume that the labeled residue was methionine. A preliminary report of these findings was presented at the I6oth Natl. Am. Chem. Soc. Meeting in Chicago in September, 197o (ref. 12). ANDERTON1 has recently reported that iodoaeetamide utilized as a site specific reagent leads to the modification of two essential histidine residues in porcine malate dehydrogenase. These residues were identified by thin-layer chromatography as well as amino acid analysis. The essential features of the reaction were identical w i t h Biochim. Biophys. Acta, 243 (x97 I) 489-497

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those of these authors 2 with respect to number of moles 14C incorporated and specificity of reaction. The differing feature in ANDERTON'S work was the use of a proteolytic digestion and an acid hydrolysis to prepare the samples for amino acid analysis and thin-layer chromatography. To corroborate ANDERTON'S findings, the E14ClCAM-histidine standard, the E14C~CAM-methionine standard and the 14C-labeled malate dehydrogenase were subjected to acid hydrolysis for 24 h i n vacuo. The effluent stream of a Beckman 116 analyzer was diverted to a fraction collector and 2-ml fractions were collected and analyzed for radioactivity. The single radioactive peak derived from the CAM-methionine standard after acid hydrolysis eluted at 92 ml, a position corresponding to carboxymethyl homocysteine, the expected major product from the acid decomposition of CAM-methionine. The CAMhistidine standard after acid hydrolysis produced 3 peaks eluting at 24, 88, and 129 ml and corresponding to 1,3-dicarboxy methylhistidine, i-carboxymethylhistidine and 3 carboxymethylhistidine, respectively 13. The CAM-malate dehydrogenase after acid hydrolysis produced one peak eluting at 128 ml which corresponds to the elution position of 3-carboxymethylhistidine. To rule out the possibility that some residue other than histidine was labeled, but lost in the subsequent acid hydrolysis, a sample of malate dehydrogenase was labeled with iodoE14C~acetamide as described in METHODS. The incorporation of iodoE14Clacetamide into malate dehydrogenase was determined and found to be 2.2 moles per mole of malate dehydrogenase. This labeled sample was then acid hydrolyzed. 96% of the original incorporated counts were recovered after hydrolysis and 92~o of this material was eluted from the analyzer at a position coincident with 3-carboxymethylhistidine. Hence histidine appears to be the only residue modified in malate dehydrogenase under these conditions. In view of this identification, it became apparent that the proteolytic digestion of labeled malate dehydrogenase carried out as described above must have released a labeled peptide resistant to further digestion which possessed properties similar to CAM-methionine on both thin-layer chromatography and on the amino acid analyzer, a circumstance which could quite easily lead to an incorrect identification of an active center residue. In order to determine the Composition of this material, t h e labeled peptide released by the action of pronase and carboxypeptidases A and B was purified on the I5-cm column of the Beckman 116 amino acid analyzer. The purified peptide was made salt-free by chromatography on a IOO cm × 1. 5 cm Bio Gel P2 column and subjected to acid hydrolysis i n vacuo at IiO ° for 24 h. The resulting amino acid analysis of the digested material revealed t h a t the peptide was composed of 2 residues of glycine and one of carboxymethylhistidine (Table III). The structure of the tripeptide was determined with an aliquot of the material purified on the I5-cm column of the Beckman analyzer. The N-terminal amino acid of the peptide was determined b y dansylation as described b y GRAY7. Approximately IO nmoles of peptide were transferred to a small glass test tube and allowed to react with dansyl chloride (I.O mg/ml in acetone) at 37 ° for I h. After hydrolysis the dansyl derivative was identified by chromatography on a polyamide layer plate as described b y WANG8. Standards were chromatographed for dansyl-glycine and dansyl-3-carboxyBiochim. Biophys. Acta, 243 (1971) 489-497

E.M. GREGORYet al.

496 TABLE III AMINO ACID COMPOSITION OF ACTIVE CENTER PEPTIDE

T h e a c t i v e c e n t e r p e p t i d e i s o l a t e d f r o m t h e i 5 - c m c o l u m n o f t h e B e c k m a n 116 a m i n o a c i d a n a l y z e r w a s a c i d h y d r o l y z e d (6 M HC1) in vacuo f o r 24 h, I i O °. T h e n u m b e r o f r e s i d u e s p r e s e n t was determined by normalization of the carboxymethylhistidine value to I residue.

A m i n o acid*

/,moles

Carboxymethylhistidine Glycine Serine Threonine

o.o694 o. 1231 o.oo9o o.oi4 °

Residues i.oo i. 77 o. 13 o.2o

* All o t h e r a m i n o a c i d s w e r e p r e s e n t in less t h a n 0 . 0 5 r e s i d u e a m o u n t s .

methylhistidine (prepared from an authentic sample of 3-carboxymethylhistidine). The dansyl derivative, after chromatography in two directions, yielded a single fluorescent spot which was identical to the standard of dansyl-3-carboxymethylhistidine. The N-I residue was determined by the method of sequential degradation plus dansylation as described by GRAY7. Approximately IOO nmoles of the original peptide were transferred to a small glass test tube and subjected to one cycle of the E d m a n degredative procedure with phenylisothiocyanate. After cyclization and cleavage with anhydrous trifluoroacetic acid the phenylhydantoin was removed by extraction with water-saturated ethyl acetate. Approximately IO nmoles of the shortened peptide were dansylated as described above. Chromatography of this product indicated that glycine was the N-I residue of the original peptide. Since the amino acid composition of this active center peptide has been determined to be composed of 2 residues of glycine and one of CAM-histidine, additional sequence steps were not required. In view of the finding that CAM-histidine resides in the N-terminal position of this peptide, it is apparent that the peptide has the structure CAM-His-Gly-Gly. COLEMAN AND VALLEE14 have previously noted the inability of carboxypeptidase A to hydrolyze peptide bonds in model peptide substrates of the type Gly Gly. Thus it seems quite possible that a sequence of CAM-His-Gly-Gly would be unaffected by treatment with carboxypeptidase A. In view of this observation and the similarities of the properties of the peptide from malate dehydrogenase and CAM-methionine on thin-layer chromatography and on the single column analyzer, one must certainly question the advisibility of utilizing proteolytic digestion (with the proteolytic enzymes listed above) as a means of digesting or separating the components of a chemically modified protein to their constituent amino acids. Although the tripeptide has been a hinderance in the determination of the original active center residue, its structure will certianly be an aid in sequence determination of the tryptic peptide now under investigation. Work is currently in progress to determine the sequence of a larger peptide derived from tryptic digestion of the iodoacetamide modified enzyme. ACKNOWLEDGMENTS

This work was supported by Grant No. HE-I2585 from the National Heart and Biochim. Biophys. Acta, 243 (1971) 4 8 9 4 9 7

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Lung Institute, National Institutes of Health, Bethesda, Maryland. (E.M.G.) was aided by a predoctoral fellowship from National Institutes of Health and (M.S.R). was aided by a predoctoral traineeship from National Institutes of Health. REFERENCES B. H. ANDERTON, Eur. J. Biochem., 15 (i97 o) 562. E. M. GREGORY AND J . H. HARRISON, Biochem. Biophys. Res. Commun., 40 (197 o) 995. C. J. R. THORNE, Biochim. Biophys. Acta, 59 (1962) 624-633. R. G. COLEMAN, Biochem. Biophys. Res. Commun., 28 (1967) 222. H. G. GUNDLACH, STANFORD MOORE AND W. H. STEIN, J. Biol. Chem., 234 (1959) i76i, A. M. KATZ, W. J. DREYER AND C. B. ANFINSEN, J. Biol. Chem., 234 (1959) 2897. W. R. GRAY, Methods Enzymol., I I (1967) 469. K. R. WOODS AND K. T. WANG, Biochem. Biophys. Acta, 133 (1967) 369. G. PFLEIDERER AND F. AURRICHIO, Biochem. Biophys. Res. Commun., 16 (1964) 53. C. J. R. THORNE AND N. O. KAPLAN, J. Biol. Chem., 238 (1963) 1861. V. LESCOVAV AND G. PFLEIDERER, Z. Physiol. Chem., 35 (1969) 492. E. M. GREGORY, M. S. ROHRBACH AND J. H. HARRISON, Abstr. I6oth Am. Chem. Soc. Natl. Meeting, z97o, Biol. Chem., 54 (197o) • 13 A. M. CRESTFIELD, W. H. STEIN AND S. MOORE, J. Biol. Chem., 238 (1963) 2413. 14 J. E. COLEMAN AND BERT L. VALLEE, Biochemistry, i (1962) lO83. 15 J. H. HARRISON, to be published. I 2 3 4 .5 6 7 8 9 io II 12

Biochim. Biophys. Acta, 243 (1971) 489-497