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The isolation and characterization of glutamic dehydrogenase from corn leaves
ARCHIVES
OF
BIOCBEMISTRY
AND
BIOPHYSICS
62, 173-183 (1956)
The Isolation and Characterization of Glutamic Dehydrogenase from Corn Leaves William A. Bulen FTOTTS the Charles F. Kettering Foundation, Yellow Springs, Ohio; and the Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio
Received October 26, 1955
The important role of glutamic acid in the intermediary metabolism of mammalian tissue has prompted recent investigators (l-3) to crystallize and characterize the enzyme glutamic dehydrogenase which catalyzes the formation or decomposition of glutamic acid according to the equation glutamate + pyridine nucleotide ti a-ketoglutarate + NH,+ + reduced pyridine nucleotide + Hf. An equally important role has been generally ascribed to glutamic acid in the metabolism of higher plants, especially since its synthesis can serve as a port of entry of ammonia into the amino acids (4, 5). Investigations of glutamic dehydrogenase in higher plants have been largely restricted to unpurified preparations. Methylene blue or thionine reduction by glutamic acid has been observed in the presence of macerates obtained from pea seeds, cabbage and carrot roots, celery, radish, and white cabbage (6), 2-3-day-old bean and pea seedlings (7) and oat coleoptiles (8, 9). Damodaran and Nair (7) measured glutamate oxidation by oxygen uptake with a saturated ammonium sulfate precipitate from bean and pea seedlings but were unable to dialyze or otherwise purify their preparation because of rapid inactivation. Von Euler et al. (10) examined the glutamic dehydrogenase activity of a dialyzed phosphate extract of cucumber seeds using Gunther’s (11) spectrophotometric assay. These workers demonstrated the reversibility of the enzyme by 173
174
WILLIAM
A. BULEN
measuring the oxidation of DPNH’ when both ar-ketoglutarate and ammonia were added to their extract. Bonner and Wildman (12) demonstrated the reduction of thionine by glutamic acid in the presence of an undialyzed “whole cytoplasm” preparation from spinach leaves (13). This paper reports the isolation of glutamic dehydrogenase from corn leaves together with some properties of the partially purified enzyme determined spectrophotometrically. METHODS AND REAGENTS Corn plants were grown from 35 to 50 days in the greenhouse from both Farm Craft Hybrid No. 40 and unclassified seed. In general, leaves were excised about 24 in. from the stock and transported immediately to the cold room where all steps in the isolation procedure were conducted at 2°C. For storage, enzyme solutions were transferred to precooled vials and placed in a deep-freeze maintained at approximately -25°C. DPN “90,” DPNH, TPN “30,” TPNH (approx. 910/o), tris(hydroxymethyl)aminomethane (Tris), calcium phosphate gel, o-iodosobenzoic acid, and p-chloromercuribenzoic acid were obtained from the Sigma Chemical Co. The DPNH was assayed with lactic dehydrogenase after the method of Horecker and Kornberg (14) and found to be 66.1% pure. The cr-ketoglutaric acid used was the analyzed reagent obtained from the California Foundation for Biochemical Research. L-Glutamic acid was obtained from Merck and Co., Inc., and n-glutamic acid from Nutritional Biochemicals Corp. EXPERIMENTAL
AND RESULTS
Spectrophotometric Assay The procedures used in assay reactions were patterned after those developed by Olson and Anfinsen (1) for use with the liver enzyme. Conditions of the assay of the plant enzyme were obtained experimentally and were altered slightly during the course of the investigations as additional information became available. The conditions described here are those considered optimal for the leaf enzyme. The composition of each reaction mixture is given with each of the accompanying tables and figures. The assay reagents were made up in the buffer to be used, and the pH was adjusted as required either with one component of the buffer or with dilute KOH. Coenzyme and ammonia solutions were freshly prepared just before use. All reagents except buffers were kept at 0°C. 1 The following abbreviations are used: DPN, diphosphopyridine nucleotide; DPNH, reduced DPN; TPN, triphosphopyridine nucleotide; TPNH, reduced TPN; Tris, tris(hydroxymethyl)aminomethane; L, light path.
GLUTAMIC
DEHYDROGENASE
175
DPNH Oxidation. The test solution consisting of 0.5 ml. of lO+ M DPNH solution, 0.2 ml. of 0.2 M potassium a-ketoglutarate, 0.2 ml. of 1.5 M (NH&SO., , and 2.0 ml. of 0.2 M Tris buffer pH 8.15 was added to the cuvette (L = 1.0 cm.) and stirred gently. At zero time, 0.1 ml. of properly diluted enzyme was added, the solution was stirred, and extinction readings at 340 rnl.c were taken every minute for 6 min. in a Beckman model DU spectrophotometer. Readings were made relative to a reference cuvette containing all reagents except the enzyme. With the reference cuvette in the light path, the optical density was arbitrarily ajusted to 0.400. The extinction decrement during the 5-min. period following the first minute was designated -AE and was proportional to the enzyme concentration at values below 0.220 (Fig. 1). The progress of the reaction is linear with respect to time during this period. There was no measurable endogenous DPNH oxidation by any of the fractions in the absence of ar-ketoglutarate. DPN Reduction. The test solution consisted of 0.5 ml. of a DPN solution containing 1 mg. DPN ‘W”/ml., 0.2 ml. of 0.2 M potassium glutamate, 2.1 ml. of 0.2 M Tris buffer pH 8.15, and 0.2 ml. of enzyme solution added at time zero. Absorption of cr-Ketoglutarate. During preliminary experiments with
FIG. 1. Initial reaction rate as a function of enzyme concentration. Final substrate concentrations: DPNH, 1.67 X lo-’ M; a-ketoglutarate, 1.33 X 10-* M; (NH&SO, , 0.1 M; in 0.2 M Tris, pH 8.15.
176
WILLIAM
A.
BULEN
the assay by DPNH oxidation, it was observed that potassium a-ketoglutarate also absorbed at 340 rnp. The extinction coefficient at 340 ml was determined by varying the a-ketoglutarate concentration in 0.2 M potassium phosphate pH 7 from 0.01 to 0.02 M. The average calculated value from four determinations was 2.075 X lo4 sq. cm./ mole. When this value is compared to the coefficient for DPNH, 6.22 X lo6 sq. cm./mole (14), changes in optical density at 340 rnp due to the formation or utilization of cY-ketoglutarate are essentially insignificant. Specific Activity. Unit activity was arbitrarily defined as the amount of enzyme producing an optical density decrement of one (-AE = l), and specific activities were calculated as units per milligram protein. Protein determination were made using the biuret reagent of Gornall, Bardawill, and David (15). Isolation of the Enzyme The procedure used to obtain the enzyme preparation used for most of the characterization experiments is as follows: Freshly cut leaves (1.7 kg.) from 45-day-old corn plants were chilled to 2°C. and macerated by grinding in a ‘Corona” corn mill (Landers, Frary and Clark, New Britain, Conn.), and the sap was obtained by straining through two layers of cheesecloth. Five milliliters of a 0.2 M KH2P0, solution containing 1% cysteine was added to each 30 ml. of sap as soon as that quantity was obtained. The addition of cysteine significantly reduces the darkening of the protein solutions and gives approximately 14% greater activity in the first (NH4)&?04 precipitate. The pH of the sap was adjusted to 5.5 with K,HPO, , precipitation was allowed to proceed for 15 min., and the sap was centrifuged for 30 min. at 25,000 X g. Screened (NH,)$04 was added slowly with stirring to render the supernatant fluid (1025 ml.) 29 % saturated (0°C.). After standing 30 min., the mixture was centrifuged and the sediment discarded. The (NHJzS04 concentration of the supernatant fluid was increased to 48% saturation and, after standing 30 min., the mixture was again centrifuged. The sediment containing the activity was dissolved in 200 ml. of 0.2 M potassium phosphate pH 8 and assayed. Sufficient 0.2 M KH904 was added to the solution to reduce the pH to 6.6, and water was added to a final volume of 450 ml. This solution was fractionated with (NH,)$04 , and the fraction precipitating between 42 and 49% saturation was dissolved in phosphate buffer pH 8, diluted to 50 ml., and assayed. The solution was then adjusted to pH 5.6 with
GLUTAMIC
177
DEHYDROGENASE
TABLE
I
from Corn Leaves Assay reactant concentrations: DPNH, 1.67 X W4 M; a-ketoglutarate, 10-z M; (NHI)zSOd ,0.05 M; in 0.2 M potassium phosphate pH 8.0. Purification
of Glutamic
Dehydrogenase
Fraction
Total units
Protein mg.
Centrifuged extract at pH 5.5 1st (NHd)zSOc 29-48s 2nd (NHa)&O1 42-49s Calcium phosphate eluate
2200 1560 680 285
13,500 1830 364 39
1.33 X
Recovery Specific activity units/mg. protein %
0.16” 0.85 1.87 7.32
100 71 31 13
a Approximate.
0.1 N HzS04 (final volume 122 ml.), stirred gently for 15 min. with 3 g. of aged calcium phosphate gel, and centrifuged. The gel was eluted by stirring 15 min. with 20 ml. of 0.2 M potassium phosphate pH 6.4 followed by centrifugation. The supernatant fluid was dialyzed for 4 hr. with agitation against two portions of 0.05 M phosphate pH 8 cont’aining 3 1. each without loss of activity. Table I summarizes the data obtained with the various fractions. The dialyzed enzyme solution contained 1.81 mg. protein/ml. and was usually diluted seven- or eightfold before addition to assay reactions. The diluted enzyme is stable at 0°C. for a period of several hours, and the undiluted material can be stored at -25°C. for periods up to 1 year without appreciable loss of activity. Properties of the Enzyme E$ect of piY. Figure 2 shows the effect of pH on the initial reaction rate in 0.2 M Tris and 0.2 M potassium phosphate buffers. The pH of reaction mixtures was determined immediately after the 6-min. reaction period with a Beckman model G glass-electrode instrument. Since the enzyme concentration and temperature were the same in both experiments, the maximum activity values can be used as a measure of the relatilre rates of reactivity in these two buffers. The initial rate in Tris is 11% higher than that observed in phosphate. The observed optimum of pH 8.1 is slightly higher than the 7.8-8.0 value reported by Damodaran and Nair (7). The enzyme, however, showed very low activity at pH 6.6 reported by Berger and Avery (9) to be a region of high activity with some of their Arena coleoptile macerates. Coenzyme Specijicity. The specificity of plant glutamic dehydrogenase for DPN was determined by substituting TPNH for DPNH, and TPN
178
WILLIAM
A. BULEN
Trir
FIG. 2. Initial reaction rate as a function of pH. Final concentration of reactants: DPNH, 1.67 X 10-4 M; a-ketoglutarate, 1.33 X 10-* M; (NH&SO4 ,0.067 M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M buffer. Temperature 23°C.
for DPN in the two assay reactions. No measurable activity was observed with TPN in either assay. These results confirm those obtained by Adler et al. (6) with crude macerates and by van Euler et al. (10) with the cucumber seed extract. Substrate Specifiity. (a) Hydroxylamine. No changes in optical density not corrected for by corresponding changes in the blank were observed when 0.067 M hydroxylamine was substituted for ammonia in the DPNH-oxidation reaction. With ammonia present, 0.0167 M hydroxylamine inhibited enzyme activity by 50%. (b) r&~utamic Acid. Under otherwise identical conditions in which L-glutamate produced an optical density increment of 0.050 in the DPNreduction assay, n-glutamate would not give a measurable reaction. These results support reports by Adler et al. (6) and by Damodaran and Nair (7) but conflict with the results obtained by Berger and Avery (8) using Arena coleoptile macerates. E$ects of Inhibitors. (a) Sulfhydryl Reagents. Inhibition studies were conducted with p-chloromercuribenzoate, o-iodoaobenzoate, and iodo-
GLUTAMIC
DEHYDROGENASE
FIG. 3. p-Chloromercuribenzoate inhibition. DPNH, 1.67 X lO+ M; a-ketoglutarate, 1.33 X hibitor at varying concentrations; enzyme, 7 0.2 M potassium phosphate pH 7.9. Temperature
179
Final concentration of reactants: 10-Z M; (NH&SO4 ,0.067 44; inpg./ml. Final volume 3.0 ml. of 26°C.
acetate. The effect of each inhibitor was tested by including varying amounts of inhibitor in the regular assay reaction and also by incubating enzyme with inhibitor for 5 min. at room temperature prior to adding the other assay reagents and initiating the reaction with the addition of DPNH. The enzyme was not inhibited by o-iodosobenzoate or iodoacetate even when incubated with the inhibitors at concentrations of 1.5 X W3 and 3 X 1W3 M, respectively. Figure 3 shows the inhibition observed with p-chloromercuribenzoate without prior incubation at concentrations up to 2.5 X 1W3 M. Even when the enzyme was incubated with this inhibitor, the inhibition was completely reversed by adding, at the end of the incubation period, sufficient glutathione solution to give a final concentration of 0.01 M. These results indicate that the enzyme possesses sulfhydryl groups essential for its enzyme activity. (b) Cyanide. When added to the other reagents in the assay reaction, NaCN at concentrations up to 4.2 X lo-’ M did not reduce the initial reaction rate. (c) Hydroxy Acids. Unlike the glutamic carboxylase of wheat leaves (16), the corn leaf dehydrogenase is not inhibited by hydroxy acids. Neither malic nor tartaric acid caused any decrease in the initial reaction
180
WILLIAM
A. BULEN
FIG. 4. Initial reaction rate as a function of a-ketoglutarate concentration. Final concentration of reactants: DPNH, 1.67 X lo-* M; (NH&SO, , 0.067 M; a-ketoglutarate, 4.17 X lo+ to 1.33 X 10-2 M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M potassium phosphate pH 8.1. Temperature 23°C.
rate even when present in the same concentration as the a-ketoglutarate (1.33 x 10-2 M). E$ect of Substrate Concentration. The effects of the concentration of DPNH, cw-ketoglutarate, and ammonia on the initial reaction rate were determined in 0.2 M potassium phosphate pH 8.1 at 23-24°C. Figures 4, 5, and 6 are graphical presentations of the data following the Lineweaver-Burk transformation (17, 18) of the Michaelis-Menten equation. No significant nonlinearity was observed with any of the reagents over the concentration ranges investigated. The Michaelis constants (I&) obtained from the data by statistical calculation are: DPNH a-Ketoglutarate Ammonia
3.65 X 10-b M 1.51 X lo-3M 0.101 M
Dialysis AgaGut Cyanide. Nicholas et al. (19) reported that a molybdenum deficiency consistently produced lowered glutamic dehydrogenase activity in extracts of Neurosporu, indicating the possible existence of a molybdenum constituent. Despite the fact that no inhibition was observed with the leaf enzyme when 4.2 X 1W4 M NaCN was added
GLUTAMIC
K,*365
0
24
6
181
DEHYDROGENASE
Y)
X 10” M
I2
(~PNH]
I4
16
u)
20
22
2.
x 10%
FIG. 5. Initial reaction rate as a function of DPNH concentration. Final concentration of reactants: (NH&SOI , 0.067 M; a-ketoglutarate, 1.33 X 10-a M; DPNH, 1.25 X KV M to 2.33 X 10-d M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M potassium phosphate pH 8.1. Temperature 23.5”C.
I.2 -
0.2 -
FIG. 6. Initial reaction rate as a function of ammonia concentration. Final concentration of reactants: DPNH, 1.67 X W4M; a-ketoglutarate, 1.33 X 10-s M; NH,+, 0.025-0.267 M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M potassium phosphate pH 8.1. Temperature 24°C.
182
WILLIAM
A. BULEN
to the assay reagents, a portion of the enzyme from a separate isolation was dialyzed for 8 hr. against 2 1. of 0.05 M potassium phosphate pH 8 containing 1W3 M NaCN. Dialysis was conducted in a cellophane dialysis sack pretreated with glutathione (20). No significant decrease in specific activity resulted. DISCUSSION
Nitrate is the principal source of nitrogen for most species of higher plants. The investigations of the McCollum-Pratt group and others (21) on the enzymatic conversion of nitrate to ammonia together with the central role of glutamic acid in transamination reactions in plants (22) point out the significance of glutamic dehydrogenase in the enzymatic conversion of ammonia to amino nitrogen. The occurrence and nature of the enzyme in leaves is of interest since in leaves the energy requirement for this’endergonic reaction could be supplied in the light by reactions mediated by chloroplasts. Vishniac and Ochoa (23) have demonstrated that chloroplast fragments from spinach leaves will reduce both DPN and TPN and have coupled this system with a glutamic dehydrogenase preparation from liver to demonstrate glutamic acid synthesis. Some of the properties of the partially purified enzyme from corn leaves have been observed. The fact that the pH optimum and substrate specificity reported here do not agree with some of, the previous work may be ascribed to the lack of specificity of dye reduction or oxygen uptake assays together with the use of unpurified enzyme preparations. SUMMARY
The isolation of glutamic dehydrogenase from corn, leaves is described together with the conditions for its spectrophotometric assay. The partially purified enzyme has a pH optimum of ‘8.1, a coenzyme specificity for DPN, and a substrate specificity for n-glutamate. Michaelis constants for DPNH, NHd+, and cY-ketoglutarate were determined. The enzyme was not inhibited by cyanide, hydroxy acids, or o-iodosobenzoate. Inhibition by p-chloromercuribenzoate was reversed by glutathione. Activity was not decreased by dialysis against cyanide. REFERENCES 1. OLSON, J. A., AND ANFINBEN, C. B., J. Biol. Chem. 197,67 (1952). 2. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 202,841 (1953). 3. STRECEER, H. J., Arch. Biochem. and Biophys. 46, 128 (1953).
GLUTAMIC
DEHYDROGENASE
183
p. 229. Academic Press Inc., New York, 4. BONNER, J., “Plant Biochemistry,” 1950. 5. ADLER, E., Arkiv Kemi, Mineral. Geol. 12B, No. 42,7 pp. (1933). 6. ADLER, E., Das, N. B., EULER, H. VON, AND HEYMAN, U., Coopt. rend. trav. lab. Carleberg 22,15 (1938). 7. DAMC&ARAN, M., AND NAIL, K. R., Biochem. J. 32.1064 (1933). 8. BER~ER, J., AND AVERY, G. S., JR., Am. J. Botany 39,290 (1943). 9. BERGER, J., AND AVERY, G. S., JR., Am. J. Botany 31, 11 (1944). 10. EULER, H. VON, ADLER, E., GUNTHER, G., AND ELLIOT, L., Enzymologio 6, 337 (1939). 11. GUENTHER, G., Arkiu Kemi, Mineral. Gee.?. 12A, No. 23 (1937). 12. BONNER, J., AND WILDMAN, S. G., Arch. Biochem. 10, 497 (1946). 13. WILDMAN, S. G., AND BONNER, J., Arch. Biochem. 14, 381 (1947). 14. HORECKER, B. L., AND KORNBERG, A., J. Biol. Chem. 176,385 (1943). 15. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 16. WEINBEROER, P., AND CLENDENNING, K. A., Can. J. Botany 30, 755 (1952). 17. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66,658 (1934). 18. WILSON, P. W., in “Respiratory Enzymes” (H. A. Lardy, ed.). Burgess Publ. Co., Minneapolis, 1949. 19. NICHOLLS, D. J. D., NASON, A., AND MCELROY, W. D., J. Biol. Chem. 207, 341 (1964). 20. NICHOLAS, D. J. D., AND NASON, A., J. Biol. Chem. 207,353 (1954). 21. WEBSTER, G. C., Ann. Rev. Plant Physiol. 6, 43 (1955). 22. WILSON, D. G., KING, K. W., AND BURRIS, R. H., J. Biol. Chem. 208,863 (1954). 23. VISHNIAC, W., AND OCHOA, S., J. Biol. Chem. 196,76 (1952).
OF
BIOCBEMISTRY
AND
BIOPHYSICS
62, 173-183 (1956)
The Isolation and Characterization of Glutamic Dehydrogenase from Corn Leaves William A. Bulen FTOTTS the Charles F. Kettering Foundation, Yellow Springs, Ohio; and the Department of Agricultural Biochemistry, The Ohio State University, Columbus, Ohio
Received October 26, 1955
The important role of glutamic acid in the intermediary metabolism of mammalian tissue has prompted recent investigators (l-3) to crystallize and characterize the enzyme glutamic dehydrogenase which catalyzes the formation or decomposition of glutamic acid according to the equation glutamate + pyridine nucleotide ti a-ketoglutarate + NH,+ + reduced pyridine nucleotide + Hf. An equally important role has been generally ascribed to glutamic acid in the metabolism of higher plants, especially since its synthesis can serve as a port of entry of ammonia into the amino acids (4, 5). Investigations of glutamic dehydrogenase in higher plants have been largely restricted to unpurified preparations. Methylene blue or thionine reduction by glutamic acid has been observed in the presence of macerates obtained from pea seeds, cabbage and carrot roots, celery, radish, and white cabbage (6), 2-3-day-old bean and pea seedlings (7) and oat coleoptiles (8, 9). Damodaran and Nair (7) measured glutamate oxidation by oxygen uptake with a saturated ammonium sulfate precipitate from bean and pea seedlings but were unable to dialyze or otherwise purify their preparation because of rapid inactivation. Von Euler et al. (10) examined the glutamic dehydrogenase activity of a dialyzed phosphate extract of cucumber seeds using Gunther’s (11) spectrophotometric assay. These workers demonstrated the reversibility of the enzyme by 173
174
WILLIAM
A. BULEN
measuring the oxidation of DPNH’ when both ar-ketoglutarate and ammonia were added to their extract. Bonner and Wildman (12) demonstrated the reduction of thionine by glutamic acid in the presence of an undialyzed “whole cytoplasm” preparation from spinach leaves (13). This paper reports the isolation of glutamic dehydrogenase from corn leaves together with some properties of the partially purified enzyme determined spectrophotometrically. METHODS AND REAGENTS Corn plants were grown from 35 to 50 days in the greenhouse from both Farm Craft Hybrid No. 40 and unclassified seed. In general, leaves were excised about 24 in. from the stock and transported immediately to the cold room where all steps in the isolation procedure were conducted at 2°C. For storage, enzyme solutions were transferred to precooled vials and placed in a deep-freeze maintained at approximately -25°C. DPN “90,” DPNH, TPN “30,” TPNH (approx. 910/o), tris(hydroxymethyl)aminomethane (Tris), calcium phosphate gel, o-iodosobenzoic acid, and p-chloromercuribenzoic acid were obtained from the Sigma Chemical Co. The DPNH was assayed with lactic dehydrogenase after the method of Horecker and Kornberg (14) and found to be 66.1% pure. The cr-ketoglutaric acid used was the analyzed reagent obtained from the California Foundation for Biochemical Research. L-Glutamic acid was obtained from Merck and Co., Inc., and n-glutamic acid from Nutritional Biochemicals Corp. EXPERIMENTAL
AND RESULTS
Spectrophotometric Assay The procedures used in assay reactions were patterned after those developed by Olson and Anfinsen (1) for use with the liver enzyme. Conditions of the assay of the plant enzyme were obtained experimentally and were altered slightly during the course of the investigations as additional information became available. The conditions described here are those considered optimal for the leaf enzyme. The composition of each reaction mixture is given with each of the accompanying tables and figures. The assay reagents were made up in the buffer to be used, and the pH was adjusted as required either with one component of the buffer or with dilute KOH. Coenzyme and ammonia solutions were freshly prepared just before use. All reagents except buffers were kept at 0°C. 1 The following abbreviations are used: DPN, diphosphopyridine nucleotide; DPNH, reduced DPN; TPN, triphosphopyridine nucleotide; TPNH, reduced TPN; Tris, tris(hydroxymethyl)aminomethane; L, light path.
GLUTAMIC
DEHYDROGENASE
175
DPNH Oxidation. The test solution consisting of 0.5 ml. of lO+ M DPNH solution, 0.2 ml. of 0.2 M potassium a-ketoglutarate, 0.2 ml. of 1.5 M (NH&SO., , and 2.0 ml. of 0.2 M Tris buffer pH 8.15 was added to the cuvette (L = 1.0 cm.) and stirred gently. At zero time, 0.1 ml. of properly diluted enzyme was added, the solution was stirred, and extinction readings at 340 rnl.c were taken every minute for 6 min. in a Beckman model DU spectrophotometer. Readings were made relative to a reference cuvette containing all reagents except the enzyme. With the reference cuvette in the light path, the optical density was arbitrarily ajusted to 0.400. The extinction decrement during the 5-min. period following the first minute was designated -AE and was proportional to the enzyme concentration at values below 0.220 (Fig. 1). The progress of the reaction is linear with respect to time during this period. There was no measurable endogenous DPNH oxidation by any of the fractions in the absence of ar-ketoglutarate. DPN Reduction. The test solution consisted of 0.5 ml. of a DPN solution containing 1 mg. DPN ‘W”/ml., 0.2 ml. of 0.2 M potassium glutamate, 2.1 ml. of 0.2 M Tris buffer pH 8.15, and 0.2 ml. of enzyme solution added at time zero. Absorption of cr-Ketoglutarate. During preliminary experiments with
FIG. 1. Initial reaction rate as a function of enzyme concentration. Final substrate concentrations: DPNH, 1.67 X lo-’ M; a-ketoglutarate, 1.33 X 10-* M; (NH&SO, , 0.1 M; in 0.2 M Tris, pH 8.15.
176
WILLIAM
A.
BULEN
the assay by DPNH oxidation, it was observed that potassium a-ketoglutarate also absorbed at 340 rnp. The extinction coefficient at 340 ml was determined by varying the a-ketoglutarate concentration in 0.2 M potassium phosphate pH 7 from 0.01 to 0.02 M. The average calculated value from four determinations was 2.075 X lo4 sq. cm./ mole. When this value is compared to the coefficient for DPNH, 6.22 X lo6 sq. cm./mole (14), changes in optical density at 340 rnp due to the formation or utilization of cY-ketoglutarate are essentially insignificant. Specific Activity. Unit activity was arbitrarily defined as the amount of enzyme producing an optical density decrement of one (-AE = l), and specific activities were calculated as units per milligram protein. Protein determination were made using the biuret reagent of Gornall, Bardawill, and David (15). Isolation of the Enzyme The procedure used to obtain the enzyme preparation used for most of the characterization experiments is as follows: Freshly cut leaves (1.7 kg.) from 45-day-old corn plants were chilled to 2°C. and macerated by grinding in a ‘Corona” corn mill (Landers, Frary and Clark, New Britain, Conn.), and the sap was obtained by straining through two layers of cheesecloth. Five milliliters of a 0.2 M KH2P0, solution containing 1% cysteine was added to each 30 ml. of sap as soon as that quantity was obtained. The addition of cysteine significantly reduces the darkening of the protein solutions and gives approximately 14% greater activity in the first (NH4)&?04 precipitate. The pH of the sap was adjusted to 5.5 with K,HPO, , precipitation was allowed to proceed for 15 min., and the sap was centrifuged for 30 min. at 25,000 X g. Screened (NH,)$04 was added slowly with stirring to render the supernatant fluid (1025 ml.) 29 % saturated (0°C.). After standing 30 min., the mixture was centrifuged and the sediment discarded. The (NHJzS04 concentration of the supernatant fluid was increased to 48% saturation and, after standing 30 min., the mixture was again centrifuged. The sediment containing the activity was dissolved in 200 ml. of 0.2 M potassium phosphate pH 8 and assayed. Sufficient 0.2 M KH904 was added to the solution to reduce the pH to 6.6, and water was added to a final volume of 450 ml. This solution was fractionated with (NH,)$04 , and the fraction precipitating between 42 and 49% saturation was dissolved in phosphate buffer pH 8, diluted to 50 ml., and assayed. The solution was then adjusted to pH 5.6 with
GLUTAMIC
177
DEHYDROGENASE
TABLE
I
from Corn Leaves Assay reactant concentrations: DPNH, 1.67 X W4 M; a-ketoglutarate, 10-z M; (NHI)zSOd ,0.05 M; in 0.2 M potassium phosphate pH 8.0. Purification
of Glutamic
Dehydrogenase
Fraction
Total units
Protein mg.
Centrifuged extract at pH 5.5 1st (NHd)zSOc 29-48s 2nd (NHa)&O1 42-49s Calcium phosphate eluate
2200 1560 680 285
13,500 1830 364 39
1.33 X
Recovery Specific activity units/mg. protein %
0.16” 0.85 1.87 7.32
100 71 31 13
a Approximate.
0.1 N HzS04 (final volume 122 ml.), stirred gently for 15 min. with 3 g. of aged calcium phosphate gel, and centrifuged. The gel was eluted by stirring 15 min. with 20 ml. of 0.2 M potassium phosphate pH 6.4 followed by centrifugation. The supernatant fluid was dialyzed for 4 hr. with agitation against two portions of 0.05 M phosphate pH 8 cont’aining 3 1. each without loss of activity. Table I summarizes the data obtained with the various fractions. The dialyzed enzyme solution contained 1.81 mg. protein/ml. and was usually diluted seven- or eightfold before addition to assay reactions. The diluted enzyme is stable at 0°C. for a period of several hours, and the undiluted material can be stored at -25°C. for periods up to 1 year without appreciable loss of activity. Properties of the Enzyme E$ect of piY. Figure 2 shows the effect of pH on the initial reaction rate in 0.2 M Tris and 0.2 M potassium phosphate buffers. The pH of reaction mixtures was determined immediately after the 6-min. reaction period with a Beckman model G glass-electrode instrument. Since the enzyme concentration and temperature were the same in both experiments, the maximum activity values can be used as a measure of the relatilre rates of reactivity in these two buffers. The initial rate in Tris is 11% higher than that observed in phosphate. The observed optimum of pH 8.1 is slightly higher than the 7.8-8.0 value reported by Damodaran and Nair (7). The enzyme, however, showed very low activity at pH 6.6 reported by Berger and Avery (9) to be a region of high activity with some of their Arena coleoptile macerates. Coenzyme Specijicity. The specificity of plant glutamic dehydrogenase for DPN was determined by substituting TPNH for DPNH, and TPN
178
WILLIAM
A. BULEN
Trir
FIG. 2. Initial reaction rate as a function of pH. Final concentration of reactants: DPNH, 1.67 X 10-4 M; a-ketoglutarate, 1.33 X 10-* M; (NH&SO4 ,0.067 M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M buffer. Temperature 23°C.
for DPN in the two assay reactions. No measurable activity was observed with TPN in either assay. These results confirm those obtained by Adler et al. (6) with crude macerates and by van Euler et al. (10) with the cucumber seed extract. Substrate Specifiity. (a) Hydroxylamine. No changes in optical density not corrected for by corresponding changes in the blank were observed when 0.067 M hydroxylamine was substituted for ammonia in the DPNH-oxidation reaction. With ammonia present, 0.0167 M hydroxylamine inhibited enzyme activity by 50%. (b) r&~utamic Acid. Under otherwise identical conditions in which L-glutamate produced an optical density increment of 0.050 in the DPNreduction assay, n-glutamate would not give a measurable reaction. These results support reports by Adler et al. (6) and by Damodaran and Nair (7) but conflict with the results obtained by Berger and Avery (8) using Arena coleoptile macerates. E$ects of Inhibitors. (a) Sulfhydryl Reagents. Inhibition studies were conducted with p-chloromercuribenzoate, o-iodoaobenzoate, and iodo-
GLUTAMIC
DEHYDROGENASE
FIG. 3. p-Chloromercuribenzoate inhibition. DPNH, 1.67 X lO+ M; a-ketoglutarate, 1.33 X hibitor at varying concentrations; enzyme, 7 0.2 M potassium phosphate pH 7.9. Temperature
179
Final concentration of reactants: 10-Z M; (NH&SO4 ,0.067 44; inpg./ml. Final volume 3.0 ml. of 26°C.
acetate. The effect of each inhibitor was tested by including varying amounts of inhibitor in the regular assay reaction and also by incubating enzyme with inhibitor for 5 min. at room temperature prior to adding the other assay reagents and initiating the reaction with the addition of DPNH. The enzyme was not inhibited by o-iodosobenzoate or iodoacetate even when incubated with the inhibitors at concentrations of 1.5 X W3 and 3 X 1W3 M, respectively. Figure 3 shows the inhibition observed with p-chloromercuribenzoate without prior incubation at concentrations up to 2.5 X 1W3 M. Even when the enzyme was incubated with this inhibitor, the inhibition was completely reversed by adding, at the end of the incubation period, sufficient glutathione solution to give a final concentration of 0.01 M. These results indicate that the enzyme possesses sulfhydryl groups essential for its enzyme activity. (b) Cyanide. When added to the other reagents in the assay reaction, NaCN at concentrations up to 4.2 X lo-’ M did not reduce the initial reaction rate. (c) Hydroxy Acids. Unlike the glutamic carboxylase of wheat leaves (16), the corn leaf dehydrogenase is not inhibited by hydroxy acids. Neither malic nor tartaric acid caused any decrease in the initial reaction
180
WILLIAM
A. BULEN
FIG. 4. Initial reaction rate as a function of a-ketoglutarate concentration. Final concentration of reactants: DPNH, 1.67 X lo-* M; (NH&SO, , 0.067 M; a-ketoglutarate, 4.17 X lo+ to 1.33 X 10-2 M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M potassium phosphate pH 8.1. Temperature 23°C.
rate even when present in the same concentration as the a-ketoglutarate (1.33 x 10-2 M). E$ect of Substrate Concentration. The effects of the concentration of DPNH, cw-ketoglutarate, and ammonia on the initial reaction rate were determined in 0.2 M potassium phosphate pH 8.1 at 23-24°C. Figures 4, 5, and 6 are graphical presentations of the data following the Lineweaver-Burk transformation (17, 18) of the Michaelis-Menten equation. No significant nonlinearity was observed with any of the reagents over the concentration ranges investigated. The Michaelis constants (I&) obtained from the data by statistical calculation are: DPNH a-Ketoglutarate Ammonia
3.65 X 10-b M 1.51 X lo-3M 0.101 M
Dialysis AgaGut Cyanide. Nicholas et al. (19) reported that a molybdenum deficiency consistently produced lowered glutamic dehydrogenase activity in extracts of Neurosporu, indicating the possible existence of a molybdenum constituent. Despite the fact that no inhibition was observed with the leaf enzyme when 4.2 X 1W4 M NaCN was added
GLUTAMIC
K,*365
0
24
6
181
DEHYDROGENASE
Y)
X 10” M
I2
(~PNH]
I4
16
u)
20
22
2.
x 10%
FIG. 5. Initial reaction rate as a function of DPNH concentration. Final concentration of reactants: (NH&SOI , 0.067 M; a-ketoglutarate, 1.33 X 10-a M; DPNH, 1.25 X KV M to 2.33 X 10-d M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M potassium phosphate pH 8.1. Temperature 23.5”C.
I.2 -
0.2 -
FIG. 6. Initial reaction rate as a function of ammonia concentration. Final concentration of reactants: DPNH, 1.67 X W4M; a-ketoglutarate, 1.33 X 10-s M; NH,+, 0.025-0.267 M; enzyme, 8 pg./ml. Final volume 3.0 ml. of 0.2 M potassium phosphate pH 8.1. Temperature 24°C.
182
WILLIAM
A. BULEN
to the assay reagents, a portion of the enzyme from a separate isolation was dialyzed for 8 hr. against 2 1. of 0.05 M potassium phosphate pH 8 containing 1W3 M NaCN. Dialysis was conducted in a cellophane dialysis sack pretreated with glutathione (20). No significant decrease in specific activity resulted. DISCUSSION
Nitrate is the principal source of nitrogen for most species of higher plants. The investigations of the McCollum-Pratt group and others (21) on the enzymatic conversion of nitrate to ammonia together with the central role of glutamic acid in transamination reactions in plants (22) point out the significance of glutamic dehydrogenase in the enzymatic conversion of ammonia to amino nitrogen. The occurrence and nature of the enzyme in leaves is of interest since in leaves the energy requirement for this’endergonic reaction could be supplied in the light by reactions mediated by chloroplasts. Vishniac and Ochoa (23) have demonstrated that chloroplast fragments from spinach leaves will reduce both DPN and TPN and have coupled this system with a glutamic dehydrogenase preparation from liver to demonstrate glutamic acid synthesis. Some of the properties of the partially purified enzyme from corn leaves have been observed. The fact that the pH optimum and substrate specificity reported here do not agree with some of, the previous work may be ascribed to the lack of specificity of dye reduction or oxygen uptake assays together with the use of unpurified enzyme preparations. SUMMARY
The isolation of glutamic dehydrogenase from corn, leaves is described together with the conditions for its spectrophotometric assay. The partially purified enzyme has a pH optimum of ‘8.1, a coenzyme specificity for DPN, and a substrate specificity for n-glutamate. Michaelis constants for DPNH, NHd+, and cY-ketoglutarate were determined. The enzyme was not inhibited by cyanide, hydroxy acids, or o-iodosobenzoate. Inhibition by p-chloromercuribenzoate was reversed by glutathione. Activity was not decreased by dialysis against cyanide. REFERENCES 1. OLSON, J. A., AND ANFINBEN, C. B., J. Biol. Chem. 197,67 (1952). 2. OLSON, J. A., AND ANFINSEN, C. B., J. Biol. Chem. 202,841 (1953). 3. STRECEER, H. J., Arch. Biochem. and Biophys. 46, 128 (1953).
GLUTAMIC
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183
p. 229. Academic Press Inc., New York, 4. BONNER, J., “Plant Biochemistry,” 1950. 5. ADLER, E., Arkiv Kemi, Mineral. Geol. 12B, No. 42,7 pp. (1933). 6. ADLER, E., Das, N. B., EULER, H. VON, AND HEYMAN, U., Coopt. rend. trav. lab. Carleberg 22,15 (1938). 7. DAMC&ARAN, M., AND NAIL, K. R., Biochem. J. 32.1064 (1933). 8. BER~ER, J., AND AVERY, G. S., JR., Am. J. Botany 39,290 (1943). 9. BERGER, J., AND AVERY, G. S., JR., Am. J. Botany 31, 11 (1944). 10. EULER, H. VON, ADLER, E., GUNTHER, G., AND ELLIOT, L., Enzymologio 6, 337 (1939). 11. GUENTHER, G., Arkiu Kemi, Mineral. Gee.?. 12A, No. 23 (1937). 12. BONNER, J., AND WILDMAN, S. G., Arch. Biochem. 10, 497 (1946). 13. WILDMAN, S. G., AND BONNER, J., Arch. Biochem. 14, 381 (1947). 14. HORECKER, B. L., AND KORNBERG, A., J. Biol. Chem. 176,385 (1943). 15. GORNALL, A. G., BARDAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 16. WEINBEROER, P., AND CLENDENNING, K. A., Can. J. Botany 30, 755 (1952). 17. LINEWEAVER, H., AND BURK, D., J. Am. Chem. Sot. 66,658 (1934). 18. WILSON, P. W., in “Respiratory Enzymes” (H. A. Lardy, ed.). Burgess Publ. Co., Minneapolis, 1949. 19. NICHOLLS, D. J. D., NASON, A., AND MCELROY, W. D., J. Biol. Chem. 207, 341 (1964). 20. NICHOLAS, D. J. D., AND NASON, A., J. Biol. Chem. 207,353 (1954). 21. WEBSTER, G. C., Ann. Rev. Plant Physiol. 6, 43 (1955). 22. WILSON, D. G., KING, K. W., AND BURRIS, R. H., J. Biol. Chem. 208,863 (1954). 23. VISHNIAC, W., AND OCHOA, S., J. Biol. Chem. 196,76 (1952).