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Inactivation of rabbit muscle l -α-glycerophosphate dehydrogenase by N-alkylmaleimides
ARCHIVES
OF
BIOCHEMISTRY
AND
Inactivation
of Rabbit
Dehydrogenase BRUCE
M. ANDERSON;
Department
138, 66-72 (1970)
BIOPHYSICS
of Biochemistry,
Muscle
L-a-Glycerophosphate
by N-Alkylmaleimides’ SO0 JA KIM, University
AND
CHIEN-NING
of Tennessee Knoxville,
WANG
Tennessee 37916
Received December 4, 1969; accepted January 30, 1970 A series of N-alkylmaleimides was shown to inactivate effectively rabbit muscle L-or-glycerophosphate dehydrogenase at pH 7.0. The second-order rate constants determined for this inactivation process increased with increasing chain length of the maleimide derivative. The binding of NADH was shown to protect the enzyme against maleimide inactivation while the binding of NAD, N1-butylnicotinamide chloride and a variety of adenine derivatives had no such effect. Product studies of the N-ethylmaleimide-inactivated enzyme revealed the presence of essentially equal amounts of S-succinylcysteine and ethylamine indicating selective modification of cysteine residues during the inactivation process. The number of lysine and histidine residues of the enzyme did not change on modification with N-ethylmaleimide. It is suggested that the chain-length effects observed in the inactivation process is related to nonpolar interactions in the binding of maleimides to the enzyme prior to an irreversible modification of sulfhydryl groups.
The binding of NAD to rabbit muscle L-CYglycerophosphate dehydrogenase (L-glycerol S-phosphate : NAD oxidoreductase, EC 1.1.1.8 .) has been shown to involve a combination of interactions between the enzyme and different parts of the coenzyme molecule (l-3). Coenzyme-competitive inhibitors structurally related to the pyridinium ring system of NAD, such as Nl-alkylnicotinamide chlorides and n-alkylammonium chlorides were demonstrated (1, 2) to interact with a “pyridinium ring region” of the NAD-binding site of this enzyme. This region can be distinguished from an “adenosine region” at the binding site where coenzyme-competitive adenine derivatives are bound (1, 2). In studies of the binding of adenosine, adenylic acid, adenosine diphosphate, and adenosine diphosphoribose, evi1 Contribution No. 79 from the Department of Biochemistry, the University of Tennessee. This work was supported by Research Grant GB 8049 from the National Science Foundation. 2 Current address: Department of Biochemistry and Nutrition, Virginia Polytechnic Institute, Blacksburg, Virginia 24061. 66
dence was obtained to indicate interactions of the phosphate groups of these compounds with the enzyme, and led to the suggestion that the NAD-binding site of this dehydrogenase also contained a “pyrophosphate region.” The binding of coenzyme-competitive inhibitors at, the “pyridinium ring region” of the NAD-binding site was observed to be facilitated by the alkyl side chains of these inhibitors indicating that in the vicinity of the “pyridinium ring region,” the enzyme is composed of relatively nonpolar residues (1, 2). This hydrophobic region was also observed to facilitate binding of coenzymecompetitive aliphatic carboxylic acids which interact with the “pyrophosphate region” of the binding site (3). Simultaneous binding of coenzyme-competitive inhibitors that interact with different regions of the NADbinding site was demonstrated (l-3) through multiple inhibition analysis as described previously (4). Since nonpolar interactions can be of importance in the binding of coenzyme-competitive inhibitors to rabbit muscle L-CC-
L-wGLYCEROPHOSPHATE
glycerophosphate dehydrogenase, a study was initiated to investigate the possibility that such interactions could also facilitate inactivation of the enzyme by irreversibly bound inhibitors. The previously demonstrated sensitivity of L-a-glycerophosphate dehydrogenase to sulfhydryl reagents (5-9) suggested that such interactions might accompany the reactions of substituted maleimides with this enzyme. A series of N-alkylmaleimides was recently used to demonstrate nonpolar interactions in the inactivation of yeast alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1) (10) and hog kidney n-amino acid oxidase (n-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) (11) through the modification of essential sulfhydryl groups. The present study reports the interactions of these compounds with rabbit muscle L-W glycerophosphate dehydrogenase. EXPERIMENTAL
PROCEDURE
Crystalline rabbit muscle ~-or-glycerophosphate dehydrogenase (L-glycerol-3-phosphate: NAD oxidoreductase, EC 1.1.1.8) was obtained in an ammonium sulfate suspension from Sigma. Stock solutions of the enzyme were prepared fresh daily in 0.05 M Tris-HCl buffer, pH 7.0. NAD, NADH, adenylic acid, adenosine diphosphate, adenosine diphosphoribose, and L-a-glycerophosphate were also obtained from Sigma. N-Ethylmaleimide was purchased from Eastman Organic Chemicals. N-Butylmaleimide was obtained from Nutritional Biochemical Corporation. N-Pentylmaleimide, N-hexylmaleimide, N-heptylmaleimide, N-octylmaleimide, and N-nonylmaleimide were prepared according to Heitz et al. (10). iV-Butylnicotinamide chloride was prepared as previously described (12). S-Succinylcysteine, prepared by the acid hydrolysis of the product obtained from the reaction of L-cysteine with N-ethylmaleimide, was purified further by chromatography on Dowex-l-formate. The S-succinylcysteine had the same properties as those reported for the compound prepared by different methods (13-16). The ninhydrin color yield for the synthetic S-succinylcysteine, expressed as aspartic acid equivalents, was 1.07, which is in good agreement with the color yield of 1.09 reported by Smyth, Blumenfeld, and Konigsberg (15) for this compound. The n-or-glycerophosphate dehydrogenase-catalyzed oxidation of L-or-glycerophosphate was studied at 25” in 3-ml reaction mixtures containing 0.05 M Tris-HCl buffer, pH 7.85, 4.3 X 1W M
67
DEHYDROGENASE
L-or-glycerophosphate and 1.84 X 10e6 M NAD. Initial velocities were obtained by measuring the linear increase in fluorescence intensity for 1 min at 460 rnp with excitation at 368 rnp. Spectrophotofluorometric measurements were carried out at 25” in a temperature-controlled cell compartment of an Aminco-Bowman spectrophotofluorometer with a Xenon mercury lamp, Pacific photometric recording photometer Model 15 fitted with an EM1 9502 photocell, and a Mosely Autograf Model 135A XY recorder. Measurements of pH were made at 25” with a Radiometer pH meter, type PHM4c, with a G-290-B glass electrode. The amino acid analyses of L-or-glycerophosphate dehydrogenase were carried out on samples containing 2.5 mg of untreated enzyme or enzyme which had been inactivated by N-ethylmaleimide. These samples were lyophilized and hydrolyzed in 1 ml of glass-distilled constant boiling HCl at 110’ for 24, 48, and 72 hr in evacuated sealed glass tubes. The hydrolyzates were lyophilized, and the residues were taken up in 0.2 M sodium citrate, pH 2.2. Quantitative analyses of the amino acids were performed on aliquots according to the method of Spackman, Stein, and Moore (17) with a Beckman/Spinco Model 116 amino acid analyzer. Fractions containing S-succinylcysteine and ethylamine, immediately aft,er passing through the calorimeters and being recorded by the analyzer, were externally collected with an automatic fraction collector. The absorbance of these samples at 570 rn+ was then determined using a Zeiss PMQ II spectrophotometer and compared to values obtained when known quantities of S-succinylcysteine and ethylamine were added to amino acid standard mixtures and processed in an identical manner. RESULTS
The time-dependent inactivation of L-cuglycerophosphate dehydrogenase by N alkylmaleimides was studied in 3-ml reaction mixtures maintained at 10’ and containing 0.05 M Tris-HCl buffer, pH 7.0, 0.2% ethanol, 6 rg of enzyme, and varying concentrations of the various maleimide derivatives. Aliquots (0.15-ml) were removed from these reaction mixtures at timed intervals, and was measured spectroenzyme activity photofluorometrically. The N-alkylmaleimides were present in concentrations always in excess of the enzyme concentration in order to promote pseudo-first-order kinetics. Ethanol (0.2%) was present in both control and experimental reaction mixtures since
68
ANDERSON,
KIM,
ethanol was required to solubilize the longer chain maleimides. The catalytic activity of n-a-glycerophosphate dehydrogenase was observed to decrease with time of incubation with the N-alkylmaleimides. The inactivation of the enzyme by N-ethylmaleimide is shown in Fig. 1 to be a first-order process. A slow inactivation in the absence of maleimide was also observed which appeared to be related to the presence of ethanol in the reaction mixture. Pseudo-first-order rate constants were calculated from the slopes of these lines, and the values obtained for six concentrations of N-ethylmaleimide are shown plotted as a function of N-ethylmaleimide concentration in Fig. 2. The pseudo-first-order rate constant obtained by extrapolation of this line to zero N-ethylmaleimide concentration is in excellent agreement with the pseudo-first-order rate constant calculated directly from the reaction carried out in the absence of the maleimide. The second-order rate constant for the N-ethylmaleimide inactivation of the enzyme was obtained from the slope of this line. Seven N-alkylmaleimides were studied in this fashion, and all effectively inactivated I
I
,
I
I
AND
WANG
n-a-glycerophosphate dehydrogenase in a pseudo-first-order process. The second-order rate constants for the inactivation processes, as well as the concentration range of the maleimides studied are shown in Table I. The effectiveness of the N-alkylmaleimides in inactivating the enzyme increases with increasing chain length of these compounds. The relationship between the second-order rate constants and the number of carbons of the maleimide derivatives is shown in Fig. 3. Various compounds which had been shown previously to be bound selectively to the NAD-binding site of L-cY-glycerophosphate dehydrogenase (1, 18) were investigated with respect to their ability to protect the enzyme against N-ethylmaleimide inactivation. Protection was recognized as a decrease in the pseudo-first-order rate constant of I
I
i ^ 6N P *
7”
6-
I
I
I
0.5
I .o
l.0X162M NEM
TABLE
I
IO
20
30 Time
40
50
60
IminI
1. Time-dependent inactivation of L-W glycerophosphate dehydrogenase by N-ethylmaleimide at IO”. The reaction mixtures contained 0.05 M Tris-HCl buffer, pH 7.0, 0.2% ethanol, 6 rg of enzyme, and N-ethylmaleimide as indicated in a total volume of 3.0 ml. Line 1, no N-ethylmaleimide; line 2,1.67 X 1V M N-ethylmaleimide, and line 3, 1 X l(Fa M N-ethylmaleimide. Enzyme activity was measured fluorimetrically. FIG.
(Mx1021
FIG. 2. The effect of N-ethylmaleimide concentration on the first-order rate constants of L-crglycerophosphate dehydrogenase inactivation. The reaction mixtures contained 0.05 M Tris-HCl buffer, pH 7.0, 0.2oj, ethanol, 6 rg of enzyme, and varying concentrations of N-ethylmaleimide, in a total volume of 3 ml.
I 0
2.0
1.5
[N-Ethylmaleimide]
I
INACTIVATION OF L-a-GLYCEROPHOSPHATE DROQENASE RY N-ALKYLMALEIMIDES Concentration range (Ml
A’ Alkylmaleimide
Ethyl Butyl Pentyl Hexyl Heptyl octy1 Nonyl
1.67 7.0 1.67 3.5 6.0 3.3 6.7
X x X x X x X
lO--1.93 10-4-4.7 1c4-l.2 10-5-3.5 l(r6-4.8 10-e-9.7 lo-‘-2.0
X x X x X x X
DEHY-
&An-~)
10-z lo-3 UF3 10-4 1O-6 10-S lO+
2.6 11.6 37.2 142 520 1730 5500
L-wGLYCEROPHOSPHATE
t
Number
of
Carbons
of
Alkyl
Group
FIG. 3. The relationship of the logarithm of the apparent second-order rate constants to the chain length of the alkyl substituents of the maleimides.
DEHYDROGENASE
Amino acid analyses were performed on samples of native and N-ethylmaleimideinactivated enzyme which had been hydrolyzed for 24, 48, and 72 hr. Average values, or values extrapolated to account for destruction during hydrolysis, for the different amino acid residues are shown in Table III. For comparison, the number of amino acid residues determined were corrected on the basis of the enzyme containing 22 aspartic acid residues as reported by Fondy et al. (19). The modified enzyme was shown to contain 10.2 residues of X-succinylcysteine and 10.7 residues of ethylamine or a S-succinylcysteine/ethylamine ratio of 1.05. Also of interest is the observation that inactivation of the enzyme resulted in no decrease in the number of lysine and histidine TABLE PROTECTION
AGAINST
TIVATION
inactivation by a constant concentration of N-ethylmaleimide. The results of these experiments are shown in Table II. Of the compounds tested, only the reduced coenzyme afforded significant protection against the maleimide inactivation. In order to determine the functional groups involved in the maleimide inactivation of the enzyme, a product study was carried out on the N-ethylmaleimide-modified enzyme. A sample (2.5 mg) of L-cyglycerophosphate dehydrogenase was incubated in 10 ml of 0.05 M Tris-HCl buffer, pH 7.04, containing 2 X 10V3 M N-ethylmaleimide until catalytic activity was reduced to less than 2% of the starting value. The inactivated enzyme was dialyzed for 18 hr at O-4” against 1 liter of 0.05 M Tris-HCl buffer, pH 7.04, followed by a second dialysis for 10 hr against the same buffer. The sample was then dialyzed for 18- and lo-hr periods against two l-liter quantities of 0.02 M ammonium bicarbonate, pH 7.85. Three 2.4~ml aliquots of the dialyzed solution were transferred to glass combustion tubes and lyophilized. These samples were hydrolyzed in 6 N HCl and assayed for amino acid composition. Three such samples of untreated L-a-glycerophosphate dehydrogenase were prepared for amino acid analysis in the same manner.
69
OF
II
N-ETHYLMALEIMIDE
INAC-
L-LX-GLYCEROPHOSPHATE
DEHYDROGENASE” Protecting
compound
Concentration Cd
2.13 4.26 6.39 2.11 4.22 6.33 7.0 1.4 2.1 1.6 3.2 4.8 4.0 6.0 8.0 1.83 3.66 5.49
NADH
AMP
ADP
ADP-ribose
Nr-Butylnicotinamide chloride NAD
X x x x X X x x x X X X x X X X X x
% Protection
l(ra 10-B lo-6 10-S lO+ RF3 10-4 10-a 10-a RF4 W4 RF4 10-z Kr2 W2 lo-& Kr6 10-s
41.4 58.0 68.4 2.3 4.2 6.7 3.0 2.8 3.0 8.2 13 12 5.6 -3.0 3.0 -3.0 0 0
0 Inactivation of the enzyme by N-ethylmaleimide was studied at 10” in 3-ml reaction mixtures containing 0.05 M Tris-HCl, pH 7.0,0.2’% ethanol, 6 fig of enzyme, and protecting compound as indicated. The pseudo-first-order rate constant obtained in the absence of protecting compounds was 6.1 X 10-* min-r. The percentage of protection was calculated from the rate constant observed in the presence of a protecting compound (/cobs.) by the following equation: % Protection
= 100 -
(k,b../6.1
X 109
X 100
70
ANDERSON, TABLE
AMINO
ACID
III
COMPOSITION
OF
NATIVE
AND
RABBIT
N-ETHYLMALEIMIDE-INaCTIVATED MUSCLE
KIM,
L-wGLYCEROPHOSPH.~TE DEHYDRO~ENASE Residues
Amino
acid
Lysine Histidine Arginine Ethylamine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteic acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine S-Succinylcysteine
based on aspartic
NdiW ~IlZYlIl~ (Fondy el al., ref1 19)
21.7 7.0 7.0 22.0 10.7 8.3 33.1 12.0 29.6 25.0 8.8 25.0 6.7 22.0 24.2 3.3 11.7 -
acid
= 22
Native enzyme
Inactivated enzynle
21.6 7.2 6.2 22.0 10.2 8.5 33.5 12.7 30.1 24.8 23.9 6.8 20.8 23.2 3.1 11.8 -
22.2 7.2 6.3 10.7 22.0 10.4
10.9 33.1 11.7 31.0 24.1 23.7 6.9 20.5 22.6 3.6 11.7 10.2
residues, two amino acids also known to react with maleimides under certain conditions. DISCUSSION
A series of N-alkylmaleimides have been shown to inactivate rabbit muscle L-W glycerophosphate dehydrogenase. This inactivation was carried out at 10” with maleimide concentrations held in excess of the enzyme, and pseudo-first-order kinetics was obtained (Fig. 1). Second-order rate constants determined for the various maleimides used (Table I) demonstrate that the rate of inactivation of the enzyme increases with increasing chain length of the alkyl substituent of the maleimide. Such a chainlength effect is not to be expected on the basis of the reactivity of these maleimides alone. Heitz et al. (10) observed no chainlength effect in the reactions of N-ethylmaleimide and N-heptylmaleimide with cysteine and glutathione. Chain-length
AND
WANG
effects observed previously in the inactivation of yeast alcohol dehydrogenase (10) and hog kidney n-amino acid oxidase (11) by these compounds were attributed to nonpolar interactions that facilitate the binding of these maleimides to these enzymes. In these cases, enzyme inactivation was suggested to occur through a two-step process, the first step being a reversible binding of the maleimide in a hydrophobic region of the enzyme and the second step being an irreversible reaction of the bound maleimide with a functional group of importance in the catalytic functioning of the enzyme. In the inactivation of n-amino acid oxidase (II), this mechanism of inactivation was more clearly indicated since the same maximum rate of inactivation was approached by saturating the enzyme with maleimides of varying chain length. In the present study, the chain-length effects on the inactivation of L-a-glycerophosphate dehydrogenase again suggest the prior binding of maleimides in the inactivation process; however, attempts to reach saturation of this enzyme with high concentrations of maleimides were unsuccessful due to the limited water solubility of these compounds. As an alternate explanation, the chainlength-dependent inactivation of L-a-glycerophosphate dehydrogenase might be attributed to a chain-length-induced denaturation or dissociation of the enzyme after a selective binding of the longer chain N-alkylmaleimides. However, in previous studies (2) of the selective binding of alkylamines, concentrations of the longer chain derivatives comparable to those used in the present study of N-alkylmaleimides did not produce a time-dependent inactivation of the enzyme. It has been shown that maleimides can react with functional groups of proteins other than the sulfhydryl groups of cysteine residues (20-22) and for this reason, the inactivation of L-a-glycerophosphate dehydrogenase was carried out under conditions where reactions with other functional groups were minimized. Such precaution is not in itself sufhcient to guarantee a specificity of maleimide reactions toward sulfhydryl groups as witnessed by a recent report (23) of a maleimide reaction resulting in a selective modification of a lysine residue
L-WGLYCEROPHOSPHATE
of ox liver glutamic dehydrogenase (Lglutamate: NAD (P) oxidoreductase (deaminating), EC 1.4.1.3) under conditions where one might expect reactions of sulfhydryl groups to predominate. One, therefore, cannot be certain of the protein functional groups reacting with maleimides unless one studies the products formed in these reactions. Such product studies have been used previously to demonstrate selective reactions of maleimides with sulfhydryl groups of a number of enzymes (11, 24-26). The product study carried out on N-ethylmaleimide-inactivated L-a-glycerophosphate dehydrogenase demonstrated the presence of essentially equal amounts of S-succinylcysteine and ethylamine after acid hydrolysis of the modified protein (Table III). This stoichiometry indicates selective modification of sulfhydryl groups of the enzyme during the inactivation process. Amino acid analyses of the untreated and maleimideinactivated enzyme show that lysine and histidine residues remain unchanged in the inactivation process. Good agreement was observed in the amino acid composition determined and the values reported previously by Fondy et al. (19); however, maleimide modification of the enzyme indicated 10 cysteine residues in the enzyme whereas cysteic acid determinations (19) had demonstrated only 9 cysteine residues present. In any case, it would appear that N-ethylmaleimide modifies all of the cysteine residues during the inactivation process. The chain-length effects observed in the inactivation of the enzyme by maleimides suggests that the sulfhydryl group or groups of importance in the catalytic activity of the enzyme reside in a nonpolar region of the and experiments are currently protein, underway to investigate the possibility that longer chain maleimides modify fewer cysteine residues in the inactivation process. In the coenzyme-competitive inhibition of L-a-glycerophosphate dehydrogenase by N’alkylnicotinamide chlorides (1) and nalkylamines (2), interactions of the positively charged nitrogens of these compounds have a directing influence on the selective binding of these compounds to the enzyme. It was originally thought that the negatively charged group of the enzyme of importance
DEHYDROGENASE
71
in these interactions was an ionized cysteine residue. The inability of Nl-butylnicotinamide chloride and NAD to protect the enzyme against inactivation by N-ethylmaleimide (Table II) argues against this possibility. Also of interest is the inability of adenine derivatives to protect the enzyme against maleimide inactivation. The excellent protection against maleimide inactivation afforded by the binding of NADH to the enzyme indicates a difference in the binding processes of the oxidized and reduced coenzymes. Differences in the binding of NAD and NADH to yeast alcohol dehydrogenase were reported previously (27). The hydrophobic regions of the coenzymebinding sites of yeast alcohol dehydrogenase and rabbit muscle L-a-glycerophosphate dehydrogenase are conveniently located to interact with the dihydronicotinamide ring of the reduced coenzyme. Evidence for such an interaction with the yeast enzyme has been obtained (27). It is felt that the binding of the dihydronicotinamide ring of the reduced coenzyme in a nonpolar region would facilitate the direct hydride transfer known to occur in reactions catalyzed by dehydrogenases. In the case of L-a-glycerophosphate dehydrogenase, the effective protection by NADH of the maleimide inactivation which is influenced by nonpolar interactions, suggests that the same hydrophobic region of the enzyme may be involved in NADH and maleimide binding. An induced conformational change on the binding of NADH cannot be ruled out as an explanation of NADH protection. In this respect, it is of interest that the binding of a major portion of the NADH molecule, such as adenosine diphosphoribose, does not elicit a similar response. REFERENCES 1. KIM, S. J., AND ANDERSON, B. M., J. Biol. Chem. 243, 3351 (1968). 2. KIM, S. J., AND ANDERSON, B. M., Biochem. Pharmacol., 17. 2413 (1968). 3. KIM, S. J., AND ANDERSON, B. M., J. Biol. Chem. 244, 231 (1969). 4. YONETANI, T., AND THEORELL, H., Arch. Bio&em. Biophys. 106, 243 (1964). 5. VAN EYS, J., NUENKE, B. J., AND PATTERSON, M. K., J. Biol. Chem., 234, 2308 (1959).
72
ANDERSON,
KIM,
6. ANKEL, H., BUCHER, TH., AND CZOK, R., Biothem. Z. 333, 315 (1960). 7. VAN EYS, J., KRETSZCHMAR, R., TSENG, N. S., AND CUNNINGHAM, L. W., JR., Biochem. Biophys. Res. Commun. 8, 243 (1962). 8. TELEGDI, M., AND KELETI, T., Acta Physiol. Acad. Sci. Hung. 26, 181 (1964). 9. HARTMAN, F. C., Fed. Proc. 27, 454 (1968). 10. HEITZ, J. R., ANDERSON, C. D., AND ANDERSON, B. M., Arch. Biochem. Biophys. 12’7, 627 (1968). 11. FONDA, M. L., AND ANDERSON, B. M., J. Biol. Chem. 244, 666 (1969). 12. ANDERSON, B. M., REYNOLDS, M. L., AND ANDERSON, C. D., Biochim. Biophys. Acta 99, 46 (1965). 13. MORGAN, E. J., AND FRIEDMANN, E., Biochem. J. 32, 733 (1938). 14. PINE, L., AND PEACOCK, C. L., J. Amer. Chem. sot. 77,3153 (1955). 15. SMYTH, D. G., BLUMENFELD, 0. O., AND KoNIGSBERG, W., Biochem. J. 91, 589 (1964). 16. KUWAKI, T., AND MIZUHARA, S., Biochim. Biophys. Acta 116, 491 (1966).
AND WANG
17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1959). 18. KIM, S. J., AND ANDERSON, B. M., J. Biol. Chem. 244, 1547 (1969). 19. FONDY, T. P., Ross, C. R., AND SOLLOHUB, S. J., J. Biol. Chem. 244, 1631 (1969). 20. SMYTH, D. G., NAGAMATSU, A., AND FRUTON, J. S., J. Amer. Chem. Sot. 82, 4600 (1960). 21. GUIDOTTI, G., AND KONIGSBERG, W., J. Biol. Chem. 239, 147 (1964). 22. BREWER, C. F., AND RIEHM, J. P., Anal. Biothem. 18, 248 (1967). 23. HOLBROOK, J. J., AND JECKEL, R., Biochem. J. 111, 689 (1969). 24. GOLD, A. H., AND SEGAL, H. L. Biochemistry 3, 778 (1964). 25. GOLD, A. H., AND SEGAL, H. L., Biochemistry 4, 1506 (1965). 26. TSUNODA, J. N., AND YASUNOBU, K. T., J. Biol. Chem. 241, 4610 (1966). 27. FONDA, M. L., AND ANDERGON, B. M., Arch. Biochem. Biophys. 120, 49 (1967).
OF
BIOCHEMISTRY
AND
Inactivation
of Rabbit
Dehydrogenase BRUCE
M. ANDERSON;
Department
138, 66-72 (1970)
BIOPHYSICS
of Biochemistry,
Muscle
L-a-Glycerophosphate
by N-Alkylmaleimides’ SO0 JA KIM, University
AND
CHIEN-NING
of Tennessee Knoxville,
WANG
Tennessee 37916
Received December 4, 1969; accepted January 30, 1970 A series of N-alkylmaleimides was shown to inactivate effectively rabbit muscle L-or-glycerophosphate dehydrogenase at pH 7.0. The second-order rate constants determined for this inactivation process increased with increasing chain length of the maleimide derivative. The binding of NADH was shown to protect the enzyme against maleimide inactivation while the binding of NAD, N1-butylnicotinamide chloride and a variety of adenine derivatives had no such effect. Product studies of the N-ethylmaleimide-inactivated enzyme revealed the presence of essentially equal amounts of S-succinylcysteine and ethylamine indicating selective modification of cysteine residues during the inactivation process. The number of lysine and histidine residues of the enzyme did not change on modification with N-ethylmaleimide. It is suggested that the chain-length effects observed in the inactivation process is related to nonpolar interactions in the binding of maleimides to the enzyme prior to an irreversible modification of sulfhydryl groups.
The binding of NAD to rabbit muscle L-CYglycerophosphate dehydrogenase (L-glycerol S-phosphate : NAD oxidoreductase, EC 1.1.1.8 .) has been shown to involve a combination of interactions between the enzyme and different parts of the coenzyme molecule (l-3). Coenzyme-competitive inhibitors structurally related to the pyridinium ring system of NAD, such as Nl-alkylnicotinamide chlorides and n-alkylammonium chlorides were demonstrated (1, 2) to interact with a “pyridinium ring region” of the NAD-binding site of this enzyme. This region can be distinguished from an “adenosine region” at the binding site where coenzyme-competitive adenine derivatives are bound (1, 2). In studies of the binding of adenosine, adenylic acid, adenosine diphosphate, and adenosine diphosphoribose, evi1 Contribution No. 79 from the Department of Biochemistry, the University of Tennessee. This work was supported by Research Grant GB 8049 from the National Science Foundation. 2 Current address: Department of Biochemistry and Nutrition, Virginia Polytechnic Institute, Blacksburg, Virginia 24061. 66
dence was obtained to indicate interactions of the phosphate groups of these compounds with the enzyme, and led to the suggestion that the NAD-binding site of this dehydrogenase also contained a “pyrophosphate region.” The binding of coenzyme-competitive inhibitors at, the “pyridinium ring region” of the NAD-binding site was observed to be facilitated by the alkyl side chains of these inhibitors indicating that in the vicinity of the “pyridinium ring region,” the enzyme is composed of relatively nonpolar residues (1, 2). This hydrophobic region was also observed to facilitate binding of coenzymecompetitive aliphatic carboxylic acids which interact with the “pyrophosphate region” of the binding site (3). Simultaneous binding of coenzyme-competitive inhibitors that interact with different regions of the NADbinding site was demonstrated (l-3) through multiple inhibition analysis as described previously (4). Since nonpolar interactions can be of importance in the binding of coenzyme-competitive inhibitors to rabbit muscle L-CC-
L-wGLYCEROPHOSPHATE
glycerophosphate dehydrogenase, a study was initiated to investigate the possibility that such interactions could also facilitate inactivation of the enzyme by irreversibly bound inhibitors. The previously demonstrated sensitivity of L-a-glycerophosphate dehydrogenase to sulfhydryl reagents (5-9) suggested that such interactions might accompany the reactions of substituted maleimides with this enzyme. A series of N-alkylmaleimides was recently used to demonstrate nonpolar interactions in the inactivation of yeast alcohol dehydrogenase (alcohol: NAD oxidoreductase, EC 1.1.1.1) (10) and hog kidney n-amino acid oxidase (n-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3) (11) through the modification of essential sulfhydryl groups. The present study reports the interactions of these compounds with rabbit muscle L-W glycerophosphate dehydrogenase. EXPERIMENTAL
PROCEDURE
Crystalline rabbit muscle ~-or-glycerophosphate dehydrogenase (L-glycerol-3-phosphate: NAD oxidoreductase, EC 1.1.1.8) was obtained in an ammonium sulfate suspension from Sigma. Stock solutions of the enzyme were prepared fresh daily in 0.05 M Tris-HCl buffer, pH 7.0. NAD, NADH, adenylic acid, adenosine diphosphate, adenosine diphosphoribose, and L-a-glycerophosphate were also obtained from Sigma. N-Ethylmaleimide was purchased from Eastman Organic Chemicals. N-Butylmaleimide was obtained from Nutritional Biochemical Corporation. N-Pentylmaleimide, N-hexylmaleimide, N-heptylmaleimide, N-octylmaleimide, and N-nonylmaleimide were prepared according to Heitz et al. (10). iV-Butylnicotinamide chloride was prepared as previously described (12). S-Succinylcysteine, prepared by the acid hydrolysis of the product obtained from the reaction of L-cysteine with N-ethylmaleimide, was purified further by chromatography on Dowex-l-formate. The S-succinylcysteine had the same properties as those reported for the compound prepared by different methods (13-16). The ninhydrin color yield for the synthetic S-succinylcysteine, expressed as aspartic acid equivalents, was 1.07, which is in good agreement with the color yield of 1.09 reported by Smyth, Blumenfeld, and Konigsberg (15) for this compound. The n-or-glycerophosphate dehydrogenase-catalyzed oxidation of L-or-glycerophosphate was studied at 25” in 3-ml reaction mixtures containing 0.05 M Tris-HCl buffer, pH 7.85, 4.3 X 1W M
67
DEHYDROGENASE
L-or-glycerophosphate and 1.84 X 10e6 M NAD. Initial velocities were obtained by measuring the linear increase in fluorescence intensity for 1 min at 460 rnp with excitation at 368 rnp. Spectrophotofluorometric measurements were carried out at 25” in a temperature-controlled cell compartment of an Aminco-Bowman spectrophotofluorometer with a Xenon mercury lamp, Pacific photometric recording photometer Model 15 fitted with an EM1 9502 photocell, and a Mosely Autograf Model 135A XY recorder. Measurements of pH were made at 25” with a Radiometer pH meter, type PHM4c, with a G-290-B glass electrode. The amino acid analyses of L-or-glycerophosphate dehydrogenase were carried out on samples containing 2.5 mg of untreated enzyme or enzyme which had been inactivated by N-ethylmaleimide. These samples were lyophilized and hydrolyzed in 1 ml of glass-distilled constant boiling HCl at 110’ for 24, 48, and 72 hr in evacuated sealed glass tubes. The hydrolyzates were lyophilized, and the residues were taken up in 0.2 M sodium citrate, pH 2.2. Quantitative analyses of the amino acids were performed on aliquots according to the method of Spackman, Stein, and Moore (17) with a Beckman/Spinco Model 116 amino acid analyzer. Fractions containing S-succinylcysteine and ethylamine, immediately aft,er passing through the calorimeters and being recorded by the analyzer, were externally collected with an automatic fraction collector. The absorbance of these samples at 570 rn+ was then determined using a Zeiss PMQ II spectrophotometer and compared to values obtained when known quantities of S-succinylcysteine and ethylamine were added to amino acid standard mixtures and processed in an identical manner. RESULTS
The time-dependent inactivation of L-cuglycerophosphate dehydrogenase by N alkylmaleimides was studied in 3-ml reaction mixtures maintained at 10’ and containing 0.05 M Tris-HCl buffer, pH 7.0, 0.2% ethanol, 6 rg of enzyme, and varying concentrations of the various maleimide derivatives. Aliquots (0.15-ml) were removed from these reaction mixtures at timed intervals, and was measured spectroenzyme activity photofluorometrically. The N-alkylmaleimides were present in concentrations always in excess of the enzyme concentration in order to promote pseudo-first-order kinetics. Ethanol (0.2%) was present in both control and experimental reaction mixtures since
68
ANDERSON,
KIM,
ethanol was required to solubilize the longer chain maleimides. The catalytic activity of n-a-glycerophosphate dehydrogenase was observed to decrease with time of incubation with the N-alkylmaleimides. The inactivation of the enzyme by N-ethylmaleimide is shown in Fig. 1 to be a first-order process. A slow inactivation in the absence of maleimide was also observed which appeared to be related to the presence of ethanol in the reaction mixture. Pseudo-first-order rate constants were calculated from the slopes of these lines, and the values obtained for six concentrations of N-ethylmaleimide are shown plotted as a function of N-ethylmaleimide concentration in Fig. 2. The pseudo-first-order rate constant obtained by extrapolation of this line to zero N-ethylmaleimide concentration is in excellent agreement with the pseudo-first-order rate constant calculated directly from the reaction carried out in the absence of the maleimide. The second-order rate constant for the N-ethylmaleimide inactivation of the enzyme was obtained from the slope of this line. Seven N-alkylmaleimides were studied in this fashion, and all effectively inactivated I
I
,
I
I
AND
WANG
n-a-glycerophosphate dehydrogenase in a pseudo-first-order process. The second-order rate constants for the inactivation processes, as well as the concentration range of the maleimides studied are shown in Table I. The effectiveness of the N-alkylmaleimides in inactivating the enzyme increases with increasing chain length of these compounds. The relationship between the second-order rate constants and the number of carbons of the maleimide derivatives is shown in Fig. 3. Various compounds which had been shown previously to be bound selectively to the NAD-binding site of L-cY-glycerophosphate dehydrogenase (1, 18) were investigated with respect to their ability to protect the enzyme against N-ethylmaleimide inactivation. Protection was recognized as a decrease in the pseudo-first-order rate constant of I
I
i ^ 6N P *
7”
6-
I
I
I
0.5
I .o
l.0X162M NEM
TABLE
I
IO
20
30 Time
40
50
60
IminI
1. Time-dependent inactivation of L-W glycerophosphate dehydrogenase by N-ethylmaleimide at IO”. The reaction mixtures contained 0.05 M Tris-HCl buffer, pH 7.0, 0.2% ethanol, 6 rg of enzyme, and N-ethylmaleimide as indicated in a total volume of 3.0 ml. Line 1, no N-ethylmaleimide; line 2,1.67 X 1V M N-ethylmaleimide, and line 3, 1 X l(Fa M N-ethylmaleimide. Enzyme activity was measured fluorimetrically. FIG.
(Mx1021
FIG. 2. The effect of N-ethylmaleimide concentration on the first-order rate constants of L-crglycerophosphate dehydrogenase inactivation. The reaction mixtures contained 0.05 M Tris-HCl buffer, pH 7.0, 0.2oj, ethanol, 6 rg of enzyme, and varying concentrations of N-ethylmaleimide, in a total volume of 3 ml.
I 0
2.0
1.5
[N-Ethylmaleimide]
I
INACTIVATION OF L-a-GLYCEROPHOSPHATE DROQENASE RY N-ALKYLMALEIMIDES Concentration range (Ml
A’ Alkylmaleimide
Ethyl Butyl Pentyl Hexyl Heptyl octy1 Nonyl
1.67 7.0 1.67 3.5 6.0 3.3 6.7
X x X x X x X
lO--1.93 10-4-4.7 1c4-l.2 10-5-3.5 l(r6-4.8 10-e-9.7 lo-‘-2.0
X x X x X x X
DEHY-
&An-~)
10-z lo-3 UF3 10-4 1O-6 10-S lO+
2.6 11.6 37.2 142 520 1730 5500
L-wGLYCEROPHOSPHATE
t
Number
of
Carbons
of
Alkyl
Group
FIG. 3. The relationship of the logarithm of the apparent second-order rate constants to the chain length of the alkyl substituents of the maleimides.
DEHYDROGENASE
Amino acid analyses were performed on samples of native and N-ethylmaleimideinactivated enzyme which had been hydrolyzed for 24, 48, and 72 hr. Average values, or values extrapolated to account for destruction during hydrolysis, for the different amino acid residues are shown in Table III. For comparison, the number of amino acid residues determined were corrected on the basis of the enzyme containing 22 aspartic acid residues as reported by Fondy et al. (19). The modified enzyme was shown to contain 10.2 residues of X-succinylcysteine and 10.7 residues of ethylamine or a S-succinylcysteine/ethylamine ratio of 1.05. Also of interest is the observation that inactivation of the enzyme resulted in no decrease in the number of lysine and histidine TABLE PROTECTION
AGAINST
TIVATION
inactivation by a constant concentration of N-ethylmaleimide. The results of these experiments are shown in Table II. Of the compounds tested, only the reduced coenzyme afforded significant protection against the maleimide inactivation. In order to determine the functional groups involved in the maleimide inactivation of the enzyme, a product study was carried out on the N-ethylmaleimide-modified enzyme. A sample (2.5 mg) of L-cyglycerophosphate dehydrogenase was incubated in 10 ml of 0.05 M Tris-HCl buffer, pH 7.04, containing 2 X 10V3 M N-ethylmaleimide until catalytic activity was reduced to less than 2% of the starting value. The inactivated enzyme was dialyzed for 18 hr at O-4” against 1 liter of 0.05 M Tris-HCl buffer, pH 7.04, followed by a second dialysis for 10 hr against the same buffer. The sample was then dialyzed for 18- and lo-hr periods against two l-liter quantities of 0.02 M ammonium bicarbonate, pH 7.85. Three 2.4~ml aliquots of the dialyzed solution were transferred to glass combustion tubes and lyophilized. These samples were hydrolyzed in 6 N HCl and assayed for amino acid composition. Three such samples of untreated L-a-glycerophosphate dehydrogenase were prepared for amino acid analysis in the same manner.
69
OF
II
N-ETHYLMALEIMIDE
INAC-
L-LX-GLYCEROPHOSPHATE
DEHYDROGENASE” Protecting
compound
Concentration Cd
2.13 4.26 6.39 2.11 4.22 6.33 7.0 1.4 2.1 1.6 3.2 4.8 4.0 6.0 8.0 1.83 3.66 5.49
NADH
AMP
ADP
ADP-ribose
Nr-Butylnicotinamide chloride NAD
X x x x X X x x x X X X x X X X X x
% Protection
l(ra 10-B lo-6 10-S lO+ RF3 10-4 10-a 10-a RF4 W4 RF4 10-z Kr2 W2 lo-& Kr6 10-s
41.4 58.0 68.4 2.3 4.2 6.7 3.0 2.8 3.0 8.2 13 12 5.6 -3.0 3.0 -3.0 0 0
0 Inactivation of the enzyme by N-ethylmaleimide was studied at 10” in 3-ml reaction mixtures containing 0.05 M Tris-HCl, pH 7.0,0.2’% ethanol, 6 fig of enzyme, and protecting compound as indicated. The pseudo-first-order rate constant obtained in the absence of protecting compounds was 6.1 X 10-* min-r. The percentage of protection was calculated from the rate constant observed in the presence of a protecting compound (/cobs.) by the following equation: % Protection
= 100 -
(k,b../6.1
X 109
X 100
70
ANDERSON, TABLE
AMINO
ACID
III
COMPOSITION
OF
NATIVE
AND
RABBIT
N-ETHYLMALEIMIDE-INaCTIVATED MUSCLE
KIM,
L-wGLYCEROPHOSPH.~TE DEHYDRO~ENASE Residues
Amino
acid
Lysine Histidine Arginine Ethylamine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Cysteic acid Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine S-Succinylcysteine
based on aspartic
NdiW ~IlZYlIl~ (Fondy el al., ref1 19)
21.7 7.0 7.0 22.0 10.7 8.3 33.1 12.0 29.6 25.0 8.8 25.0 6.7 22.0 24.2 3.3 11.7 -
acid
= 22
Native enzyme
Inactivated enzynle
21.6 7.2 6.2 22.0 10.2 8.5 33.5 12.7 30.1 24.8 23.9 6.8 20.8 23.2 3.1 11.8 -
22.2 7.2 6.3 10.7 22.0 10.4
10.9 33.1 11.7 31.0 24.1 23.7 6.9 20.5 22.6 3.6 11.7 10.2
residues, two amino acids also known to react with maleimides under certain conditions. DISCUSSION
A series of N-alkylmaleimides have been shown to inactivate rabbit muscle L-W glycerophosphate dehydrogenase. This inactivation was carried out at 10” with maleimide concentrations held in excess of the enzyme, and pseudo-first-order kinetics was obtained (Fig. 1). Second-order rate constants determined for the various maleimides used (Table I) demonstrate that the rate of inactivation of the enzyme increases with increasing chain length of the alkyl substituent of the maleimide. Such a chainlength effect is not to be expected on the basis of the reactivity of these maleimides alone. Heitz et al. (10) observed no chainlength effect in the reactions of N-ethylmaleimide and N-heptylmaleimide with cysteine and glutathione. Chain-length
AND
WANG
effects observed previously in the inactivation of yeast alcohol dehydrogenase (10) and hog kidney n-amino acid oxidase (11) by these compounds were attributed to nonpolar interactions that facilitate the binding of these maleimides to these enzymes. In these cases, enzyme inactivation was suggested to occur through a two-step process, the first step being a reversible binding of the maleimide in a hydrophobic region of the enzyme and the second step being an irreversible reaction of the bound maleimide with a functional group of importance in the catalytic functioning of the enzyme. In the inactivation of n-amino acid oxidase (II), this mechanism of inactivation was more clearly indicated since the same maximum rate of inactivation was approached by saturating the enzyme with maleimides of varying chain length. In the present study, the chain-length effects on the inactivation of L-a-glycerophosphate dehydrogenase again suggest the prior binding of maleimides in the inactivation process; however, attempts to reach saturation of this enzyme with high concentrations of maleimides were unsuccessful due to the limited water solubility of these compounds. As an alternate explanation, the chainlength-dependent inactivation of L-a-glycerophosphate dehydrogenase might be attributed to a chain-length-induced denaturation or dissociation of the enzyme after a selective binding of the longer chain N-alkylmaleimides. However, in previous studies (2) of the selective binding of alkylamines, concentrations of the longer chain derivatives comparable to those used in the present study of N-alkylmaleimides did not produce a time-dependent inactivation of the enzyme. It has been shown that maleimides can react with functional groups of proteins other than the sulfhydryl groups of cysteine residues (20-22) and for this reason, the inactivation of L-a-glycerophosphate dehydrogenase was carried out under conditions where reactions with other functional groups were minimized. Such precaution is not in itself sufhcient to guarantee a specificity of maleimide reactions toward sulfhydryl groups as witnessed by a recent report (23) of a maleimide reaction resulting in a selective modification of a lysine residue
L-WGLYCEROPHOSPHATE
of ox liver glutamic dehydrogenase (Lglutamate: NAD (P) oxidoreductase (deaminating), EC 1.4.1.3) under conditions where one might expect reactions of sulfhydryl groups to predominate. One, therefore, cannot be certain of the protein functional groups reacting with maleimides unless one studies the products formed in these reactions. Such product studies have been used previously to demonstrate selective reactions of maleimides with sulfhydryl groups of a number of enzymes (11, 24-26). The product study carried out on N-ethylmaleimide-inactivated L-a-glycerophosphate dehydrogenase demonstrated the presence of essentially equal amounts of S-succinylcysteine and ethylamine after acid hydrolysis of the modified protein (Table III). This stoichiometry indicates selective modification of sulfhydryl groups of the enzyme during the inactivation process. Amino acid analyses of the untreated and maleimideinactivated enzyme show that lysine and histidine residues remain unchanged in the inactivation process. Good agreement was observed in the amino acid composition determined and the values reported previously by Fondy et al. (19); however, maleimide modification of the enzyme indicated 10 cysteine residues in the enzyme whereas cysteic acid determinations (19) had demonstrated only 9 cysteine residues present. In any case, it would appear that N-ethylmaleimide modifies all of the cysteine residues during the inactivation process. The chain-length effects observed in the inactivation of the enzyme by maleimides suggests that the sulfhydryl group or groups of importance in the catalytic activity of the enzyme reside in a nonpolar region of the and experiments are currently protein, underway to investigate the possibility that longer chain maleimides modify fewer cysteine residues in the inactivation process. In the coenzyme-competitive inhibition of L-a-glycerophosphate dehydrogenase by N’alkylnicotinamide chlorides (1) and nalkylamines (2), interactions of the positively charged nitrogens of these compounds have a directing influence on the selective binding of these compounds to the enzyme. It was originally thought that the negatively charged group of the enzyme of importance
DEHYDROGENASE
71
in these interactions was an ionized cysteine residue. The inability of Nl-butylnicotinamide chloride and NAD to protect the enzyme against inactivation by N-ethylmaleimide (Table II) argues against this possibility. Also of interest is the inability of adenine derivatives to protect the enzyme against maleimide inactivation. The excellent protection against maleimide inactivation afforded by the binding of NADH to the enzyme indicates a difference in the binding processes of the oxidized and reduced coenzymes. Differences in the binding of NAD and NADH to yeast alcohol dehydrogenase were reported previously (27). The hydrophobic regions of the coenzymebinding sites of yeast alcohol dehydrogenase and rabbit muscle L-a-glycerophosphate dehydrogenase are conveniently located to interact with the dihydronicotinamide ring of the reduced coenzyme. Evidence for such an interaction with the yeast enzyme has been obtained (27). It is felt that the binding of the dihydronicotinamide ring of the reduced coenzyme in a nonpolar region would facilitate the direct hydride transfer known to occur in reactions catalyzed by dehydrogenases. In the case of L-a-glycerophosphate dehydrogenase, the effective protection by NADH of the maleimide inactivation which is influenced by nonpolar interactions, suggests that the same hydrophobic region of the enzyme may be involved in NADH and maleimide binding. An induced conformational change on the binding of NADH cannot be ruled out as an explanation of NADH protection. In this respect, it is of interest that the binding of a major portion of the NADH molecule, such as adenosine diphosphoribose, does not elicit a similar response. REFERENCES 1. KIM, S. J., AND ANDERSON, B. M., J. Biol. Chem. 243, 3351 (1968). 2. KIM, S. J., AND ANDERSON, B. M., Biochem. Pharmacol., 17. 2413 (1968). 3. KIM, S. J., AND ANDERSON, B. M., J. Biol. Chem. 244, 231 (1969). 4. YONETANI, T., AND THEORELL, H., Arch. Bio&em. Biophys. 106, 243 (1964). 5. VAN EYS, J., NUENKE, B. J., AND PATTERSON, M. K., J. Biol. Chem., 234, 2308 (1959).
72
ANDERSON,
KIM,
6. ANKEL, H., BUCHER, TH., AND CZOK, R., Biothem. Z. 333, 315 (1960). 7. VAN EYS, J., KRETSZCHMAR, R., TSENG, N. S., AND CUNNINGHAM, L. W., JR., Biochem. Biophys. Res. Commun. 8, 243 (1962). 8. TELEGDI, M., AND KELETI, T., Acta Physiol. Acad. Sci. Hung. 26, 181 (1964). 9. HARTMAN, F. C., Fed. Proc. 27, 454 (1968). 10. HEITZ, J. R., ANDERSON, C. D., AND ANDERSON, B. M., Arch. Biochem. Biophys. 12’7, 627 (1968). 11. FONDA, M. L., AND ANDERSON, B. M., J. Biol. Chem. 244, 666 (1969). 12. ANDERSON, B. M., REYNOLDS, M. L., AND ANDERSON, C. D., Biochim. Biophys. Acta 99, 46 (1965). 13. MORGAN, E. J., AND FRIEDMANN, E., Biochem. J. 32, 733 (1938). 14. PINE, L., AND PEACOCK, C. L., J. Amer. Chem. sot. 77,3153 (1955). 15. SMYTH, D. G., BLUMENFELD, 0. O., AND KoNIGSBERG, W., Biochem. J. 91, 589 (1964). 16. KUWAKI, T., AND MIZUHARA, S., Biochim. Biophys. Acta 116, 491 (1966).
AND WANG
17. SPACKMAN, D. H., STEIN, W. H., AND MOORE, S., Anal. Chem. 30, 1190 (1959). 18. KIM, S. J., AND ANDERSON, B. M., J. Biol. Chem. 244, 1547 (1969). 19. FONDY, T. P., Ross, C. R., AND SOLLOHUB, S. J., J. Biol. Chem. 244, 1631 (1969). 20. SMYTH, D. G., NAGAMATSU, A., AND FRUTON, J. S., J. Amer. Chem. Sot. 82, 4600 (1960). 21. GUIDOTTI, G., AND KONIGSBERG, W., J. Biol. Chem. 239, 147 (1964). 22. BREWER, C. F., AND RIEHM, J. P., Anal. Biothem. 18, 248 (1967). 23. HOLBROOK, J. J., AND JECKEL, R., Biochem. J. 111, 689 (1969). 24. GOLD, A. H., AND SEGAL, H. L. Biochemistry 3, 778 (1964). 25. GOLD, A. H., AND SEGAL, H. L., Biochemistry 4, 1506 (1965). 26. TSUNODA, J. N., AND YASUNOBU, K. T., J. Biol. Chem. 241, 4610 (1966). 27. FONDA, M. L., AND ANDERGON, B. M., Arch. Biochem. Biophys. 120, 49 (1967).