Purification and properties of rat brain hexokinase

ARCHIVES

OF

BIOCHEMISTRY

AND

Purification

BIOPHYSICS

and

ALBERT Biochemistry

Department, Received

161,

48-55 (1972)

Properties C. CHOU Michigan January

of Rat Brain AND

JOHN

State University, 27, 1972; accepted

Hexokinase’

E. WILSON East Lansing, March

Michigan

4882S

31, 1972

Rat brain hexokinase has been purified to homogeneity as judged by disc-gel electrophoresis, isoelectric focusing, and analytical ultracentrifugation. More than 50% of the initial activity could be obtained in homogeneous form (sp act, 60 units/mg protein) by a simple procedure consisting essentially of two steps: relatively specific solubilization of the enzyme from the mitochondrial membrane by glucose&P, followed by DEAE-cellulose column chromatography. The molecular weight is approximately 98,000; this same molecular weight was observed when the denatured enzyme was examined by the SDS-polyacrylamide electrophoretic technique, strongly suggesting that the enzyme consists of a single polypeptide chain. In accord with this view, a single N-terminal amino acid, glycine, has been recovered in 80% yield based on a molecular weight of 98,000. The amino acid composition of the rat brain hexo-

kinase has been determined and found to be very similar to that previously reported for the bovine brain enzyme (Schwartz, G. P., and Basford, R. E. (1967) Biochemistry 6,1070, suggesting extensive sequence homology. A notable feature of the brain hexokinases is a relatively low aromatic amino acid content, as judged acid composition and the relatively low molar extinction coefficient.

ATP (3, 4, 9). This latter observation has been advantageously employed in purification of the hexokinase from rat brain. The present paper describes a simple procedure for the isolation of the glucose-6phosphate-solubilized rat, brain hexokinase in high yield. The purified enzyme is homogeneous when examined by electrophoretic or ultracentrifugal methods, or by isoelectric focusing. Some chemical and physical properties of the enzyme are also reported, including amino acid composition, molecular weight, and subunit structure. A preliminary report of this work has been made (10).

The major portion of the hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) activity in brain homogenates is

bound to the mitochondria (14). Approximately half of this particulate activity is overt, that is, the activity is directly measurable with no pretreatment of the particles; the other half of the particulate activity exists in a latent form which may be rendered overt by membrane-disrupting techniques such as freeze-thaw cycles, sonication, osmotic shock, or detergents (5, 6). Current evidence suggests that the latent enzyme is located on mitochondria enclosed in the synaptosome, the synaptosomal membrane being the permeability barrier resulting in latency (7, 8). The particulate enzyme may be rather specifically eluted from the mitochondria by glucose-6-phosphate or

EXPERIMENTAL

* Abbreviations used: HEPES, N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid; BSA, bovine serum albumin; SDS, sodium dodecyl sulfate. 4s

0 1972 by Academic of reproduction in any

Press, Inc. form reserved.

PROCEDURE

Materials. Biochemicals and HEPES” buffer were obtained from Sigma Chemical Co. DEAEcellulose was purchased from Gallard-Schlesinger.

1 This research was supported in part by Grant NS 09910-01 from the National Institute of Neurological Diseases and Stroke, National Institutes of Health, United States Public Health Service.

Copyright All rights

by the amino

RAT

BRAIN

All other materials were Reagent Grade, obtained from commercial sources. Adult male rats (approximately 350-500 g) of the Sprague-Dawley type were obtained from Spartan ResearcYh (Haslett, MI) and maintained on common laboratory diet and water ad libitum. Enzyme assag. Hexokinase activity was determined spectrophotometrically at 25°C f 0.5” in an assay mixture containing 3.3 mM glucose, 6.7 mM ATP, 6.‘7 rnM MgC12, 40 mM HEPES, 10 rnM 1-thioglycerol, 0.32 mM NADP, and 1 unit of glucoseS-phosphate dehydrogenase in a total volume of 1 .O ml (pH 7.5). NADPH formation was followed at 340 nm with a Turner Model 330 Spectrophotometer connected to a Sargent SRL Recorder. With samples containing glucose-6-P, a slightly modified assay was performed: the sample was added to an assay mixture containing all components except ATP, and glucose-6-phosphate oxidation was permitted to go to completion (approximately 2-3 min). Subsequently, the hexokinase assay was initiated by addition of 0.035 (pH 7.3). One unit is ml of 0.2 M ATP solution defined as the amount of enzyme which catalyzes the formation of 1 pmole of glucose-6-P per minute. Lipoic dehydrogenase (NADHz:lipoamide oxidoreductase, EC 1.6.4.3) (11) and lactate dehydrogenase (n-1actate:NAD oxidoreductase, EC 1.1.1.27) (12) were assayed according to previously described procedures. Hemoglobin was measured by the absorbance at 405 nm. Protein determination. This was carried out by a turbidimetric micromethod as described by Katzenellenbogen and Dobryszycka (13). Crystalline BSA was used as standard. When sucrose or buffer was present in the sample analyzed, the standards were prepared in equivalent solutions. Polyacrylamide disc-gel and sodium dodecyl sulfate polyacrylamide disc-gel electrophoresis. Polyacrylamide disc-gel electrophoresis of bhe native enzyme on 7.5yo polyacrylamide gel was performed with Tris-glycine buffer, pH 8.4, essentially as described by Davis (14). The gel was subjected to electrophoresis for 40 min at 2 mA per gel and 4°C prior to sample application. Electrophoresis was carried out at 4°C using 5 mA of current per gel for 2 hr. After electrophoresis, the gels were placed in 10yo trichloroacetic acid solution for 2030 min, then stained for 3 hr with 0.4y0 Coomassie blula in 10% trichloroacetic acid-333 methanol followed by destaining in 107, trichloroacetic acid-33% methanol. The destained gel was scanned at 600 a with a Gilford Model 2410 linear-transport accessory. SDS-polyacrylamide disc-gel electrophoresis was carried out by the method of Shapiro et al. (15) as modified by Johnson et al. (16). Human

HEXOKINASE

49

r-globulin, phosphorylase A (a-1,4-glucan:orthophosphate glucosyltransferase, EC 2.4.1.1) BSA, ovalbumin, and pepsin (EC 3.4.4.1) were used as standard marker proteins. All proteins were denatured and reduced by incubation in a solution of 0.1 M sodium phosphate buffer (pH 7.1), 1% SDS, 1% 2-mercaptoethanol, and 10% glycerol. Where indicated, 7 M urea or 5.5 M guanidine hydrochloride was also added to this denaturing medium. The incubation was performed at 60°C for 6 hr or 100°C for 40 min. Ten microliters of bromphenol blue (0.057,) was added to each protein preparation to serve as a migration marker during the electrophoresis. The electrophoresis buffer contained 0.1 M sodium phosphate, pH 7.1, and 0.170 SDS. Electrophoresis was performed at room temperature and a constant current of 8 mA per gel, and the gels were stained as described above except that the staining period was extended to lo-14 hr. The staining and destaining processes were performed in the dark, a precaution said to enhance the sensitivity of the staining procedure with SDS-gels (J. Johnson, personal communication). Electrofocusing. The electrofocusing column (LKB 8101, LKB Instruments) was prepared according to the manufacturer’s directions with the pH 5-8 ampholine. The enzyme was dialyzed against 1% glycine solution for 4 hr before applying to the column. The initial voltage setting was 300 V and this was increased to 600 V after 4 hr; focusing was complete at 48 hr. The temperature was maintained at 2°C throughout the experiment. Fifty-two fractions were collected for protein and activity assay. Sucrose density-gradient centrijugation. The procedure used for sucrose density-gradient centrifugation followed that of Martin and Ames (17). Linear sucrose gradients of 5-20yo sucrose, phosphate (pH containing 0.01 M potassium 7.0), 0.01 M glucose, and 0.5 mM EDTA were prepared. A sample, containing purified hexokinase and marker proteins in a volume of 0.1 ml was carefully applied to the top of the gradient. Hemoglobin, lipoic dehydrogenase, and lactic dehydrogenase were used as marker proteins. Centrifugation was at 40,000 rpm for 18 hr at 4°C in a Spinco Model L Centrifuge with a SW-50.1 rotor. After centrifugation, contents of the tube were collected in lo-drop fractions and assayed for hexokinase and marker proteins. Ultracentrifugal analysis. Sedimentation equilibrium ultracentrifugation was performed in a Spinco Model E ultracentrifuge equipped with Rayleigh interference optics. The exact temperature was obtained with a calibrated rotor and temperature indicator control (RTIC) unit. Spectroscopic II-G plates were measured with a

50

CHOU AND WILSON

Bausch and Lomb microcomparator. The molecular weight was determined by the meniscusdepletion technique of Yphantis (18). The enzyme, 0.41 mg/ml, was centrifuged at 2°C and 20,410 rpm for 48 hr in 0.05 M potassium phosphate-O.1 M KC1 buffer (pH 7.0). The data were analyzed with the CDC 3600 computer (Michigan State University Computer Center) using a program previously described (19). The output of the computer included the molecular weight f standard deviation. A partial specific volume of 0.74 ml/mg was calculated from the amino acid composition (20). The authors gratefully acknowledge the invaluable assistance of Dr. George Stance1 in carrying out the ultracentrifuge experiments. Amino acid analysis. Samples were hydrolyzed with constant boiling HCl at 110°C for 24, 48, or 72 hr. A few crystals of phenol were added to the vial prior to evacuation and sealing to aid in preventing decomposition during hydrolysis (D. C. Robertson, personal communication). Analysis of the hydrolyzate was kindly performed by Mrs. Diana Ersfeld using an analyzer designed by Dr. Donald C. Robertson in collaboration with Dr. W. A. Wood. Minor corrections (
Puri$cation

of rat brain hexokinase. Rat

brains (45 g) were frozen in liquid nitrogen immediately after removal, a procedure previously shown to expose the latent activity (6, 9). After thawing, the brains were

homogenized in 450 ml cold 0.25 M sucrose solution (10 ml/g brain) using a Teflonglass homogenizer (Size C, A. H. Thomas

Co.). The homogenate was centrifuged at 1OOOg X 20 mm at 4°C and the pellet discarded. The supernat.ant fraction was centrifuged at 40,OOOy X 20 min at 4°C. After discarding the supernatant fluid, the resulting pellet was washed three times by suspending in 450 ml of 0.25 M sucrose solution followed by centrifugation at 40,OOOg X 20 min. The final pellet was resuspended in 900 ml of 0.25 M sucrose solution (20 ml/g brain). The suspension was made 1 mM in glucose6-P by addition of 0.01 volume of a 0.1 >i solution (pH 7.0 & 0.2) and incubated at 25°C for 1 hr with occasional gentle stirring. After incubation, the suspension was centrifuged at 105,OOOgfor 1 hr at 10°C. The supernatant fluid, which contained solubilized hexokinase, was decanted and potassium phosphate buffer (1 M, pH 7.0), glucose (1 M), Na*EDTA (50 mM) and 1-thioglycerol (12 M) were added to a final concentration of 0.01 M, 0.01 M, 0.5 m&I, and 2 mM, respectively. The supernatant fluid (918 ml) was then concentrated to about 21 ml by use of an Amicon ultrafiltration device with a PM-10 membrane. In general, 1 g of fresh brain provides about 7 units of solubilized HK with a specific activity of approximately 15 units/mg. The concentrated extract was loaded onto a DEAE-cellulose column (2 X 25 cm), previously equilibrated with 10 mu potassium phosphate buffer, pH 7.0, containing 10 mM glucose, 0.5 m&r Na2EDTA, and 2 mM 1-thioglycerol, and the column washed with this buffer overnight. A linear 600-ml gradient from 0 to 0.3 ~RIKC1 in this buffer was used to elute hexokinase. Fractions of 4.8 ml were collected at a flow rate of 29 ml per hour. The enzyme was eluted at a KC1 concentration of 0.06 M. The elution profile is presented in Fig. 1. Approximately 84% of the activity was recovered in the peak fractions, which were pooled and concentrated to about 5 ml. The concentrated sample was then dialyzed exhaustively against 10 mM glucose+.5 MM EDTA-2 mM thioglycerol-10 ~LV potassium phosphate (pH 7.0) to remove an unidentified, nonprotein contaminant which had been eluted from the DEAE-

RAT

BRAIN

HEXOKINASE

51

I 7-

04- -0.20

6E'

FRACrfON

1. DEAE-cellulose column chromatography of rat brain hexokinase. Chromatography was performed as described in the text. Protein (Am) in the effluent was followed with an Isco Model UA-2 monitor, and the dotted curve (. . .) as a tracing of this record. KC1 concentration (0) was determined by conductivity measurements on the fractions. FIG.

column in a broad peak (O-O.15 M KCl). Solid n-glucose was added to a final concentration of 0.2 M and the enzyme stored at -20°C. No loss of activity occurred over a period of several months, even after repeated freezing and thawing. Results of a typical purification are shown in Table I. Examination of homogeneity by electrophoresisand isoelectricfocusing. The enzyme was homogeneous on the basis of polyacrylamide disc gel electrophoretic and isoelectrofocusing studies. Analytical polyacrylamide disc-gel electrophoresis of up to 0.4 mg of the purified hexokinase indicated a single protein band (Fig. 2). Isoelectrofocusing revealed a single activity peak which coincided with the protein peak (Fig. 3) ; 75 % of the initial hexokinase activity was recovered after focusing. The isoelectric point of the purified enzyme is 6.35. Molecular weight. The molecular weight of purified hexokinase was determined by analytical ultracentrifugation and sucrose density-gradient centrifugation. The results of an analytical ultracentrifugal experiment are shown in Fig. 4. The excellent agreement of the data points with a theo-

TABLE PURIFICATION

OF RAT

I BRAIN

Pro-

Fraction

YYm

tein bw)

Original homogenate 46,066g pellet, washed 3X Glucose-6-P solubilized DEAB-column eluate Concentrated eluate

465

-LI

HEXOKINASE %i ac-

Activity (units’ ) (

tivity NJ/ w)

1

--

915

-

-

481 ( 100)

-

232

48

-

918

19.1

295

61

15.4

62

4.1

248

52

66.5

4.0

243

51

60.5

4.8

= The particulate nature of the enzyme at this stage precluded use of the turbidimetric protein assay employed in this work.

retically expected straight line (18) attests to the hydrodynamic homogeneity of the preparation. Calculation of the molecular weight from these data gave a value of 97,500 f 500 (SD). The results of sucrose density-gradient centrifugation experiments are presented in Fig. 5. Plotting the sedimentation velocity

52

CHOU AND WILSON

1)

T

0.2A

I . 1 5 :

Mig..tim+ FIG. 2. Electrophoretic homogeneity of purified rat brain hexokinase. A sample (100 pg) of the purified enzyme was electrophoresed, and the gel was stained as described in the text. The inset shows the actual gel. The densimetric tracing obtained by scanning the gel at 600 nm is shown above. The slight upturns at either end of the curve represent the top (cathode) and bottom (anode) of the gel. Increasing the sample to 400 pg did not show any additional bands.

of the marker proteins against the molecular weights to the two-third power gave the expected linear relationship (17), from which a molecular weight of 98,000 was found for hexokinase, in excellent agreement with the value obtained by the sedimentation equilibrium method. Xubunzt structure. The subunit structure of the purified enzyme was examined by the SDS-polyacrylamide disc-gel electrophoresis technique. After denaturation for 6 hr at 6O”C, the enzyme moved as a single band with a mobility corresponding to a molecular weight of 96,000-98,000. The plot of migration of protein versus logarithm of molecular weight for purified hexokinase and for standard proteins is shown in Fig. 6. The inclusion of 7 M urea or 5.5 M guanidine hydrochloride in the denaturing medium, or denaturing the enzyme at 100°C for 40 min gave identical results, as did the use of 7 % acrylamide instead of 4.25 % acrylamide gels. These results strongly suggest that rat brain hexokinase consists of a single polypeptide chain with a molecular weight of approximately 98,000. Amino acid composition and X-terminal amino acid. A partial amino acid composition of the purified enzyme is shown in Table II where it is compared with that of the bovine enzyme reported by Schwartz

.lO .9 .a 7 .6 c 5 4 3

.-

--

--

.-

FRACTJON

FIG. 3. Isoelectric focusing of purified rat brain hexokinase. The conditions of the experiment were as described in the text. Thirty units (0.5 mg) of purified enzyme were used; 75% of the initial activity was recovered after focusing. Activity (0); protein, (A); PH (0).

RAT

BRAIN

HEXOKINASE

21 0.1

0.2

0.3

0*

0.5

0.6

Mobility

I 51.5

L

50.0

51.0

50.5 r=t

cdl

FIG. 4. Analytical ultracentrifugation of purified rat brain hexokinase. The plot is according to Yphantis (18) ; y0 - y is the fringe displacement (proportional to protein concentration) and r is the radius of rotation. The conditions of the experiment and analysis of results were as described in the text. The molecular weight calculated from the slope is 97,500 f 509 (SD). L.<,i. D”

r.oo1500

2000 iMolecular

FIG. 6. SDS-polyacrylamide-gel electrophoresis of purified rat brain hexokinase and marker proteins. The procedure is described in the text. From these results, a subunit molecular weight of 96,000-98,096 was determined for the hexokinase

weight)

2500 us

1 3000

FIG. 5. Molecular weight of purified rat brain hexokinase from sucrose density gradient centrifugation. The positions of the marker proteins are plotted against molecular weight to the 34 power, giving a straight-line relationship (17). From this plot, the molecular weight of hexokinase is found to be 98,000.

and Basford (23). The tryptophan content was estimated by the spectrophotometric procedure of Edelhoch (24) ; two different preparations of the enzyme gave values of 3.8 and 4.1 tryptophan residues per mole of enzyme. Sulfhydryl groups were determined essentially according to Ellman (25); 9.2 sulfhydryls per mole of enzyme were found in each of two determinations with the native enzyme in 0.1 M potassium phosphate, pH 7.6, while t’he inclusion of 5.2 M guanidine hydrochloride in the reaction medium increased t’his to 13.2 f 0.4 (average of three determinations f av. dev.). A single N-terminal amino acid was found by the dansyl procedure, and identified as glycine. In two experiments, quantitation of the N-terminal amino acid revealed 0.79 and 0.83 mole of glycine per mole of purified enzyme, based on a molecular weight of 98,000. This result also suggests that rat brain hexokinase consists of a single polypeptide chain, in agreement with the SDS electrophoresis studies. Ultraviolet absorption spectrum. The ultraviolet absorption spectrum of the purified rat brain enzyme was virtually identical to that previously reported for the bovine brain enzyme (23), with an absorption maximum at 278 nm and three distinct shoulders at approximately 269, 265, and 259 nm. The relatively low molar extinction coefficient at 280 nm, 5.1 f 0.1 X lo* cm2mole-l (average for four determinations f

54

AMINO

CHCU

ACID

TABLE II COMPOSITION OF RAT BRAIN HEXOKINASES

BOVINE

moles amino acida mole histidine

Amino acid

Rat Aspartate Threonine Serine Glutamate Proline Glycine Alanine Valine Cystine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Histidine Arginine Total residues histidine-

AND

AND

5.40 2.77 2.63 4.93 1.96 4.42 2.64 3.60 1.64 2.74 4.62 1.11 2.07 3.28 3.05 per

f f dc I: f f f f <1 f f f f f f (1.00) f 46.86

0.09 0.04 0.11 0.03 0.04 0.08 0.06

phenylalanine and tyrosine to figure more prominently in the absorption spect,rum of the enzyme (26). DISCUSSION

Bovine’ 0.01 0.01 0.03 0.07 0.07 0.17 0.04 0.07

WILSON

5.00 2.83 2.83 5.33 1.17 4.33 2.66 3.17 trace 1.67 2.33 4.83 1.00 2.17 3.17 (1 .OO) 2.50 44.99

a The results are expressed as moles amino acid per mole histidine for purposes of comparison with the ratios reported for the bovine brain enzyme. Since it is calculated that rat brain hexokinase contains 18 histidines per mole, multiplication of the ratios by 18 will give the number of each residue per mole of enzyme. 6 Results of Schwartz, G. P., and Basford, R. E. (1967) Biochemislry 6, 1070.

av. dev.)3, is compatible with the relatively low aromatic amino acid content. The appearance of the fine structure below 280 nm in the absorption spectrum may simply be due to an unusually low tryptophan content, thereby permitting the absorption due to J The absorption at 280 nm increases during frozen storage of the enzyme; this increase is accompanied by the appearance of “absorption” at longer wavelengths (e.g., 330350 nm), possibly indicating light-scattering changes due to aggregation of the enzyme. No activity loss has been observed to accompany this phenomenon, nor is there observable precipitation of the enzyme. We have not investigated this situation further. We mention it here only to point out that the extinction coefficient given must be used only under conditions where turbidimetric contributions are negligible; e.g., with freshly prepared enzyme.

Procedures for the solubilization and purification of brain hexokinase have been reported by several investigators (23,27-29). All previous procedures have utilized relatively laborious techniques to release the enzyme from the mitochondria. Furthermore, no procedure which can provide highly purified enzyme with high yield has been reported; e.g., the previously described method for bovine brain hexokinase (23) reportedly gave homogeneous enzyme, but the overall yield was only 7 % of the activity in the initial homogenate. By our method, a pure solubilized hexokinase can be easily isolated from rat brain and purified by a simple purification step with a final recovery of more than 50 %. The unique feature of the purification scheme is the relatively specific solubilization of the enzyme from the mitochondria by glucose-6-P. Maximal solubilization of particulate hexokinase occurs only after treatment of the particles by some membrane-disrupting technique (5, 6, 9). We find the freezethaw treatment with liquid nitrogen to be the most efficient and convenient method to expose the latent hexokinase of rat brain. Increasing the temperature during solubilization from 25°C to 37°C causes a slight (lo-15 %) increase in solubilization but also results in increased nonspecific solubilization of other proteins, leading to a lower specific activity (7.5 units/mg) of solubilized enzyme. Yeast hexokinase exists as a dimer of subunits having a molecular weight of approximately 50,000 (30, 31). Moreover, “light” and “heavy” forms of wheat germ hexokinase have been reported (32), suggesting possible subunit structure in this plant hexokinase. In contrast, our studies indicate that the rat brain enzyme consists of a single polypeptide chain with a molecular weight of 98,000. Easterby (33) has recently found that porcine heart hexokinase also consists of a single polypeptide chain having a molecular weight of 97,000. Thus,

RAT

BRAIN

subunit structure may be a fundamental difference between mammalian hexokinases and those from lower organisms. Three isozymes of hexokinase have been found in various rat tissues (27). In view of the observaCons indicating a single polypeptide chain structure for the type I isozyme found in brain, it seems unlikely that the isozymes of hexokinase can be explained as dimeric combinations of two basic subunit types, analogous to the situation with the lactic dehydrogenase isozymes (34). The amino acid composition of rat brain hexokinase is very similar to that previously reported by Schwartz and Basford for the bovine brain enzyme (23). Utilizing the results shown in Table II, we have calculated the Metzger difference index (35) to be 3.8, suggesting (not unexpectedly) the existence of extensive sequence homology between the rat brain and bovine brain hexokinases. Based on previous sequence comparisons using this index (36), a difference index of 3.8 suggests that more than 90% of the sequences may ‘be homologous. REFERENCES 1. CRANE, R. K., AND SOLS, A. (1953) J. Biol. Chem. 2Q3, 273. M. K. (1960) Biochemical J. 77, 2. JOHNSON, 610. I. A., AND WARMS, J. V. B. (1965) Fed. 3. ROSE, Proc. 24, 297. I. A.., AND WARMS, J. V. B. (1967) J. 4. ROSE, Biol. Chem. 242, 1635. I)., AND TEICHGRABER, P. (1967) 5. BIESOLD, Biochem. J. 103, 13C. 6. WILSON, J. E. (1967) Biochem. Biophys. Res. Commun. 28, 123. 7. WILSON, J. E., AND BARCH, D. (1971) Fed. Proc. 30, 1139. J. E., Arch. Biochem. Biophys., in 8. WILSON, press. J. E. (1968) J. Biol. Chem. 243,364O. 9. WILSON, 10. CHOTJ, A. (1971) Fed. Proc. 30, 1103. 11. MASSEY, V. (1966) Methods Enzymol. 9, 272. 12. KORNBERG, A. (1955) Methods Enzymol. 1, 441. W. M., AND DOBRYS13. K.4TZENELLENBOGEN,

HEXOKINASE ZYCKA,

xi W.

M.

(1959)

CZin.

Chim.

Acta

4,

515. 14. DAVIS, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404. 15. SHAPIRO, A. L., VIWUELA, E., AND MAIZEL, J. V. (1967) Biochem. Biophys. Res. Commun. 28, 815. J. C., DEBACKER, M., AND BOEZI, 16. JOHNSON, J. A. (1971) J. BioZ. Chem. 246,1222. 17. MARTIN, R. G., AND AMES, B. N. (1961) J. BioZ. Chem. 236, 1372. 18. YPHANTIS, D. A. (1964) Biochemistry 3, 297. 19. HAMMERSTEDT, R. H., M~~HLER, H., DECKER, K. A., AND WOOD, W. A. (1971) J. BioZ, Chem. 246, 2069. 20. SCHACHMAN, H. K. (1957) Methods Enzymol. 4, 33. 21. WOODS, K. R., AND WANG, K. T. (1967) Biochim. Biophys. Acta 133, 369. 22. WANG, K. T., AND Wu, P. H. (1968) J. Chromatogr. 37, 353. 23. SCHWARTZ, G. P., AND BASFORD, R. E. (1967) Biochemistry 6, 1070. H. (1967) Biochemistry 6, 1948. 24. EDELHOCH, 25. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70. 26. K.~WAHARA, F. S., WANG, S., AND TALALAY, P. (1962) J. BioZ. Chem. 237, 1500. 27. GROSSB~RD, L., AND SCHIMKE, R. T. (1966) J. BioZ. Chem. 241, 3546 (1966). 28. MOORE, C. L. (1968) Arch. Biochem. Biophys. 128, 734. 29. JOSHI, M. D., AND JAGANNATHAN, V. (1966) in Methods in Enzymology (Colowick, Sidney P., and Kaplan, Nathan O., eds.), Vol. 9, p. 371. J. J., AND COLOWICK, S. P. (1970) 30. SCHMIDT, Fed. Proc. 29, 334. 31. RUSTUM,

Y. M., MASSARO, E. J., AND BARE. A. (1971) Biochemistry 10, 3509. MEUNIER, J. C., But, J., .~ND RICARD, J. (1971) Fed. Eur. BioZ. Sot. Lelt. 14, 25. EASTERBY, J. S. (1971) Fed. Eur. BioZ. Sot. Lett. 18, 23. COHN, R. D., KAPLAN, N. O., LEVINE, L., AND ZWILLING, E. (1962) Science 136, 962. METZGER, H., SHAPIRO, M. B., MOSIMANN, J. E., AND VINTON, J. E. (1968) ‘Vature London 219, 1166. FONDY, T. P., AND HOLOHAN, P. D. (1971) J. Theor. BioZ. 31, 229. NARD,

32. 33. 34. 35.

36.