A GLUTATHIONE REDUCTASE FROM ESCHERICHIA COLI*

Transcription

1 A GLUTATHIONE REDUCTASE FROM ESCHERICHIA COLI* BY ROBERT E. ASKIS (From the Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelph,ia, Pennsylvania) (Received for publication, July 26, 1954) Earlier studies had demonstrated that a flavoprotein capable of mediating a triphosphopyridine nucleotide (TPN)-linked reduction of Furacin (5-nitro-2-furaldehyde semicarbazone) was present in cell-free extracts of Furacin-susceptible strains of Escherichia coli, but was absent in extracts of resistant strains (I). Dr. Philip E. Hartman, who was then associated with our department, suggested to us that the flavoprotein involved might be a TPN-linked reductase for oxidized glutathione (GSSG). The presence of such a GSSG reductase had already been demonstrated in cell-free extracts of yeast (a), plant tissues (3-5), mammalian tissues (6), and Neurospora (7), although these reductases had not been identified specifically as flavoproteins. A partially purified preparation of our Furacin-reducing flavoprotein was indeed found to contain a very active TPN-linked GSSG reductase. However, it quickly became evident that this reductase was a flavoprotein distinctly different from the Furacin-reducing one which we have been studying. Thus the latter flavoprotein was (a) absent in cell-free extracts of resistant strains, (b) after treatment with acid could be almost completely reactivated by 3 X 1CF M riboflavin phosphate (FP) but not by an equivalent concentration of flavin adenine dinucleotide (FAD), and was (c) completely destroyed by being heated for 10 minutes at 70. The GSSG reductase, on the other hand, was (a) present in amounts with high activity in extracts of both susceptible and resistant strains of E. coli, (b) could be reactivated after acid treatment by 3 X 1e6 M FAD, but not by as much as 50 times this concentration of FP, and (c) lost only 10 to 15 per cent of its activity when heated to 70 for 10 minutes. Since such treatment enabled us to achieve a relatively high degree of separation of the GSSG reductase, some further characterization studies of this enzyme were made and are here presented. Test Methods and Materials Test System-Enzymatic activity was measured with a Beckman spectrophotometer, model DU, in terms of t.he rate of decrease of opt)ical density * This work was uxulc possil,lc I)y grants from the I+::tton I,abor:ttories, Inc., Norwich, New York. 77

2 78 GLUTATHIONE REDUCTASE FROM E. COLI at the 340 rnp absorption peak for reduced TPN (TPNH). Unless otherwise specified, all the studies were carried out with final concentrations of 3.6 X lo-& M TPNH, 5.4 X 1t4 M GSSG, and M/15 phosphate buffer, ph 7.0, in a total volume of 3.0 ml. at 30. Control systems containing TPNH and enzyme but no GSSG showed no activity. Preparation of EnzI/me-Crude cell-free extracts of E. coli, strain ECFS, were prepared as previously described for the partial purification of glycerol dehydrogenase up to the step at which the extracts used to prepare the dehydrogenase were heated to for 90 minutes (8). At this step removal of most of the nucleoproteins was accomplished by the following modification of a method described by Korkes and his coworkers (9). A volume of 1.0 M manganous chloride equal to 0.05 the volume of the crude extract was added slowly to it at 3-5 during constant stirring, and the extract was held at 3-5 for an additional 30 minutes with frequent stirring. The precipitated material was then removed by centrifugation for 20 minutes at 10,000 r.p.m. (12,800 X g) in a Servall angle centrifuge, and the supernatant fluid was dialyzed overnight at 3-5 against about 15 times its volume of distilled water. Further precipitation occurred during this dialysis. Solid ammonium sulfate was added to the dialyzed material to 40 per cent saturation, and the precipitate was removed by centrifugation in the Servall centrifuge for 20 minutes at 10,000 r.p.m. and discarded. The supernatant solution was now brought to 60 per cent saturation with solid ammonium sulfate and centrifuged as before and the supernatant liquid discarded. The precipitate was dissolved in a volume of distilled mater equal to half the original volume of the extract, heated to 70 for 10 minutes in a water bath, and rapidly chilled to 3, and the heat-coagulated protein was removed by centrifugation as described above. Solid ammonium sulfate was added to the supernatant fluid to 60 per cent saturation at 3 and the material recentrifuged. The precipitate was again dissolved in a volume of cold (3-5 ) distilled water equal to one-half the volume of the original crude extract. This solution was then dialyzed twice for 4 hours at 3-5 against a solution containing 0.1 M phosphate buffer, ph 7.0, and M cysteine 30 times it.s volume. The above procedure resulted in an approximately 50-fold purification of the enzyme in terms of TPNH oxidized per mg. of protein nitrogen. The best preparations had a specific activity of 2.6 in terms of micrograms per ml. of total protein required to produce a decrease of in the optical density reading of the spectrophotometer at 340 rnp in the 1st minute. Such partially purified preparat,ions did not oxidize either reduced diphosphopyridine nucleotide (DPNH) or TPNH at ph 7.0 in the presence of cystine, 02, triphenyltetrazolium chloride, neotetrazolium chloride, cytochrome c, or Furacin. In t,he presence of TP?I T-I, sodium 2,&dichloro-

3 R. E. ASNIS 79 benzenoneindophenol was reduced at a measurable but relatively slow rate. There was no measurable oxidation of DPNH in the presence of enzyme and GSSG. These preparations were quite stable and could be kept in the refrigerator at 4 for several weeks with only slight loss of activity. They were best preserved in the frozen state, since freezing resulted in no loss of activity. It should be noted here that omission of glucose from the media in which the E. coli cells were grown greatly decreased the amount of GSSG reductase present in the cell-free extracts. Thus extracts of cells grown in media without glucose had 50 to 60 per cent less GSSG reductase activity. Interestingly enough, there was no measurable decrease in the amount of the previously mentioned TPN-linked Furacin-reducing flavoprotein in extracts of cells grown without glucose. Acid Treatment of GSSG Reductase was carried out by the following modification of the method of Warburg and Christian (10). A given volume of the flavoprotein was chilled to 1 and held at this temperature. There was added during constant stirring an equal volume of cold (3 ) saturated ammonium sulfate solution which had been acidified to ph 0.6 with concentrated hydrochloric acid. This preparation was held at 1 and vigorously stirred for 10 minutes, after which it was centrifuged for 10 minutes at 10,000 r.p.m. (10,500 X g) in a Spinco refrigerated centrifuge. The supernatant fluid was discarded and the precipitate redissolved in a volume of cold (3 ) ~/15 phosphate buffer, ph 7.0, equal to the original volume of enzyme preparation used. TPNH was prepared from 80 per cent TPN 80 (Sigma). For most of the experiments the TPN was reduced with hydrosulfite by the following modification of the method of Ohlmeyer for reduction of DPN (11). 10 mg. of TPN were dissolved in 5 ml. of a solution containing 1.0 per cent sodium bicarbonate and 0.1 per cent sodium hydrosulfite. This solution was allowed to stand for 2 hours, after which it was vigorously aerated for 30 minutes. By this method 90 to 95 per cent of the theoretical yield of TPNH was obtained. Such chemically reduced TPN was used in all of the experiments except those involving iodometric titration of GSH and the reduction of sodium 2, G-dichlorobenzenoneindophenol. Apparently traces of either sodium hydrosulme or sodium sulfite present. in these TPNH preparations caused a non-enzymatic reduction of iodine and of sodium dirhlorobenzenoneindophenol. Therefore, in all experiment s involving these two reagents, TPKH was prepared enzymatically with glucose-b-phosphate and a preparation of glucose-6-phosphate dehydrogenase. Aft,er enzymatic reduction of TPN was complete, the dehydrogenase was inactivated by heating of the preparation in a boiling water bath for 5 minutes.

4 80 GLUTATHIONE REDUCTASE FROM E. COLI DPNH was prepared from chromatographically pure DPN (Schwarz Laboratories) by the hydrosulfite method described for TPNH. GSSG and GSH (sodium salt) were commercial preparations. FAD used in preliminary experiments was a 15 per cent preparation, FAD 15 (Sigma). For the final experiment, shown in Fig. 4, Dr. D. J I TIME IN MINUTES FIG. 1. First order reaction rates of the TPN-linked enzymatic reduction of GSSG. a = initial concentration of TPNH, z = decrease in concentration of TPNH in a given time, 0 = 4.6 -y per ml. of final total protein concentration, 0 = 2.3 y per ml. of final total protein concentration, 0 = 1.15 y per ml. of final total protein concentration, A = 0.58 y per ml. of final total protein concentration. I O Kane kindly gave us some 80 per cent FAD, which had been prepared in the laboratories of Dr. I. C. Gunsalus. FP was a commercial synthetic preparation. Quantitative determination of GSH was made by titration with N iodine in acid solution (3). Protein Determination-The protein content of the cell-free enzyme preparations was estimated turbidimetrically after precipitation with trichloroacetic acid (12).

5 R. E. ASNIS 81 Results Enxymatic Activity-In the presence of GSSG the enzyme mediated oxidation of TPNH at a rate indicative of a first order reaction (Fig. 1). That the rate of oxidation of TPNH is a function of enzyme concentration is illustrated in Fig. 2. Stoichiometrically an approximate 1:2 molar rela L PROTEIN CONCENTRATION t&g x IO- /ML) FIG. 2. Relationship of enzyme concentration in terms of final protein concentration. Optical density readings taken 1 minute after t,he start of each reaction. tionship between TPNH oxidized and GSH produced was demonstrated by the following: TPNH oxidized, 0.708,0.375,0.927 pmole; GSH produced, 1.608, 0.790, pmoles, respectively, by titration with N 12. This is in agreement with the schematic equation TI NII + H+ + GSSG $ TPN+ + 2GSH The concentrations for optimal activity (V,,,,,.) were approximately 1.2 X 1W4 mole per liter for TPNH and approximately 3.3 X 1CY3 mole

6 82 GLUTATHIONE REDUCTASE FROM E. COLI per liter for GSSG. The K, for the reductase and TPNH was found to be 3.7 X 1O-5 mole per liter; the K, for the reductase and GSSG was found to be 1.4 X 1O-3 mole per liter. As previously reported (3, 6), the apparent equilibrium point of the reaction is greatly in favor of the formation of GSH. Thus when final concentrations of 4.3 X 10e4 M GSH and 6.7 X 1e5 M TPN were used over a ph range from 6.0 to 10.0, there was never any detectable reduction of TPN PH FIG. 3. Relationship of GSSG reductase activity to ph. Optical density readings taken 1 minute after the start of the reaction. Biochemical Properties qf Enzyme-The ph range for activity and the ph optimum of approximately 6.9 are shown in Fig. 3. That the enzyme is a flavoprotein is evident from its inactivation by acid treatment and subsequent reactivation by the addition of FAD (Fig. 4). That FAD is probably the natural prosthetic group is indicated by the fact that resolved preparations could not measurably be reactivated by molar concentrations of FP or riboflavin 50 times the active concentration of FAD used (Fig. 4). It should be noted here that there was no measurable non-enzymatic oxidation of TPNH at ph 7.0 in the presence of GSSG and 3 X 1V M FAD. By concentrating the total yield of partially purified flavoprotein from 24 liters of culture into 3.0 ml., it was possible to analyze it spectrophotometrically. There was strong absorption in the

7 R. E. ASNIS 83 ultraviolet region with a maximum at 267 to 268 mp. In the visible region there was only one small but unmistakable maximum at 450 rnp. The effect of some familiar enzyme inhibitors and three antibiotics on the reductase was investigated, and the results are shown in Table I. As can n w N 0 x 0 3 I z a I- -f 0 2 x H 2 I 0 I 2 TIME IN MINUTES FIG. 4. Reactivation of acid-treated reductase by 3 X lowe M FAD. 0 = untreated enzyme, X = acid-treated enzyme + 3 X 10m6 M FAD, A = acid-treated enzyme, A = acid-treated enzyme X lo- M FP, A = acid-treated enzyme X lo- M riboflavin. be seen, the reductase was relatively resistant to the effects of several metals known to inhibit certain enzymes and was highly resistant to cyanide (KCN) and to the antibiotics used. Sodium p-chloromercuribenzoate completely inhibited it, in a final concentration of M; this inhibition could be completely reversed by the addition of M cysteine, indicating that the reductase is dependent on sulfhydryl groups for its activity. There was no evidence for the presence of any essential metal component

8 84 GLUTATHIONE REDUCTASE FROM E. COLI in the reductase. Thus no loss in activity occurred when 5.0 ml. of the partially purified preparation were dialyzed overnight at 3-5 against 1000 ml. of a solution containing 0.1 M phosphate buffer, ph 8.2, 0.01 M KCN, and M cysteine. Moreover, as can be seen in Table I, 0.1 M KCN (a strong metal-binding agent) had no measurable inhibitory effect on the enzyme. As already indicated in the method for its partial purification, the reductase is relatively heat-stable. When held at 65 for 10 minutes it showed no loss in activity. 10 minutes at 70 caused only a 10 to 15 per cent loss of activity. However, 10 minutes at 80 resulted in complete loss of activity. TABLE Effect of Various Enzywle Inhibitors and Antibiotics on, GSSG Reductase Activity Compound Sodium p-chloromercuribenzoate ( cd-+ (cuso~) < Zni Fe- (ZnSOa) (FeCI;%). tested I Final molar concentration of compound Per cent inhibition KCN (0.1 M), penicillin (0.01 M), Chloromycetin ( M), Aureomycin ( M), and Furacin ( M) showed no inhibition. DISCUSSION The presence of a highly active GSSG reductase in cell-free extracts of E. coli is not surprising in view of its wide distribution, as shown by earlier investigators (2-7). It is even less surprising in the light of recent studies demonstrating that cells of E. coli contain large amounts of GSH (13). GSH apparently has many functions in life processes (14-16). That the GSSG reductase has proved to be a flavoprotein is in agreement with the fact that many other enzymes mediating the oxidation of reduced pyridine nucleotides have proved to be flavoproteins, such as, for example, the diaphorases, the cytochrome reductases, and nitrate reductase The author wishes to express his appreciation to Dr. Philip E. Hartman for advice, and to Miss Mary Catherine Glick and Miss Ute Busemann for technical assistance. SUMMARY 1. A highly specific, relatively heat-stable TPN-linked GSSG rcduc%ase has been isolated from a strain of 13. coli and partially purified.

9 R. E. ASNIS Reduction of 1 mole of GSSG to 2 moles of GSH for every mole of TPNH oxidized has been demonstrated. 3. The GSSG reductase has been shown to be a flavoprotein, for which FAD (but not FP or riboflavin) can serve as the prosthetic group. 4. Kinetic studies of the partially purified enzyme showed the constant K, for TPNH and for GSSG to be 3.7 X W6 and 1.4 X lws mole per liter, respectively. 5. The optimal activity was at ph 6.9. The enzyme was 100 per cent inhibited by M p-chloromercuribenzoate and this inhibition was completely reversed by M cysteine, indicating dependence of the enzyme on sulfhydryl groups for its activity. In relatively high (0.001 M) concentrations, Fe + and Zn* caused 50 per cent inhibition; Cu* at and M caused 100 and 45 per cent inhibition, respectively. KCN (0.1 M), penicillin (0.01 M), Chloromycetin ( M), Aureomycin ( M), and Furacin ( M) caused no measurable inhibition. BIBLIOGRAPHY 1. Asnis, R. E., Bact. Proc., 81 (1953). 2. Meldrum, N. U., and Tar-r, H. L. A., Biochem. J., 29,108 (1935). 3. Mapson, L. W., and Goddard, D. R., Rio&em. J., 49,592 (1951). 4. Conn, E. E., and Vennesland, B., J. Biol. Chem., 192, 17 (1951). 5. Anderson, G. A., Stafford, H. A., Corm, E. E., and Vennesland, B., Plant Physiol., 27, 675 (1952). 6. Rall, T. W., and Lehninger, A. L., J. Biol. Chem., 194,119 (1952). 7. Nicholas, D. J. D., Nason, A., and McElroy, W. D., J. Biol. Chem., 207, 341 (1954). 8. Asnis, R. E., and Brodie, A. F., J. Biol. Chem., 203, 153 (1953). 9. Korkes, S., de1 Campillo, A., Gunsalus, I. C., and Ochoa, S., J. Biol. Chem., 193, 721 (1951). 10. Warburg, O., and Christian, W., Biochem. Z., 298, 368 (1938). 11. Ohlmeyer, P., Biochem. Z., 297, 66 (1938). 12. Stadtman, E. R., Novelli, G. D., and Lipmann, F., J. Biol. Chem., 191, 365 (1951). 13. Roberts, R. B., and Bolton, E. T., Science, 115, 479 (1952). 14. Racker, E., and Krimsky, I., Nature, 169, 1043 (1952). 15. Barron, E. S. G., Texas Rep. Biol. and Med., 11, 653 (1953). 16. Racker, E., Federation Proc., 12, 711 (1953).

10 A GLUTATHIONE REDUCTASE FROM ESCHERICHIA COLI Robert E. Asnis J. Biol. Chem. 1955, 213: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at ml#ref-list-1