ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 288, No. 1, July, pp. 48-53, 1991

Inhibition of Glutathione by Glutathione’ Phyllis

M. Chung,

Roseann

E. Cappel,

Verna and Marrs McLean Department

Received November

Disulfide Reductase and Hiram

of Biochemistry,

5, 1990, and in revised form February

F. Gilbert’

Baylor College of Medicine, Houston, Texas 77030

12, 1991

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetitive inhibitor against GSSG and an uncompetitive inhibitor against NADPH at saturating concentrations of the fixed substrate. In both cases, the inhibition by GSH is parabolic, consistent with the requirement for 2 eq. of GSH in the reverse reaction. The inhibition of GSSG reduction by physiological levels of the product, GSH, would result in a significantly more oxidizing intracellular environment than would be realized in the absence of inhibition. Considering inhibition by the high intracellular concentration of GSH, the steady-state concentration of GSSG required to maintain a basal glutathione peroxidase flux of 300 nmol/min/g in rat liver is estimated at 8-9 PM, about lOOO-fold higher than the concentration of GSSG predicted from the equilibrium constant for glutathione reductase. The kinetic properties of glutathione reductase also provide a rationale for the increased glutathione (GSSG) efflux observed when cells are exposed to oxidative stress. The resulting decrease in intracellular GSH relieves the noncompetitive inhibition of glutathione reductase and results in an increased capacity (V,,,) and decreased K, for GSSG. o 1991 Academic PESS, IN.

The tripeptide, glutathione (y-glutamylcysteinylglytine) and its disulfide comprise the major, low-molecularweight thiol/disulfide redox buffer of most cells (1). Intracellular glutathione (GSH)3 levels are regulated by a complex mechanism involving control of synthesis, transport out of the cell, and utilization in transport, redox, and detoxification processes (1). While much is 1 Supported by Grant HL-28521 from the National Institutes of Health and by a summer fellowship (P.M.C.) from the Baylor College of Medicine. ’ To whom correspondence should be addressed. 3 Abbreviations used: DTT, dithiothreitol; protein-SSG, protein-glutathione mixed disulfide.

known regarding the regulation and variation of the total cellular glutathione pool (predominantly GSH), much less is known about the maintenance and regulation of the levels of glutathione disulfide (GSSG). Although the myriad reactions of GSH complicate the quantitative analysis of GSH metabolism and synthesis, GSSG appears to suffer only four metabolic fates (l-3). GSSG may be reduced to GSH in an NADPH-dependent process catalyzed by glutathione reductase, GSSG can be exported from the cell, or it may react with protein thiols generating either protein-SS-glutathione mixed disulfides or protein-SS-protein disulfides with protein dithiols (3). GSSG is produced principally by the action of the seleno-enzyme glutathione peroxidase (4), 2 GSH + H202 --* GSSG + 2 HzO.

[II

The major fate of this GSSG, at least kinetically, is reduction back to GSH, catalyzed by the NADPH-dependent flavoprotein, glutathione reductase (5, 6) (Eq. [a]), GSSG + NADPH

+ H+ = NADP+ + 2 GSH.

[2]

These two enzymes, glutathione peroxidase and glutathione disulfide reductase, comprise the glutathione peroxidase/reductase cycle which serves to detoxify a major portion of the cellular hydroperoxides generated by the metabolism of oxygen (7). There are two major obstacles to understanding the regulation and maintenance of cellular GSSG levels-the difficulty in measuring low levels of GSSG without significant interference by GSH autoxidation (8), and the effects of intracellular compartmentation. Experiments employing suitable controls and analytical procedures suggest that the total cellular level of GSSG in rat liver amounts to about 0.2-0.3s of the level of GSH ([GSH]/ [GSSG] = 300-400) (2,3,9,10). This puts the intracellular concentration of GSSG in the range of 0.03-0.05 mM assuming a uniform distribution of GSSG throughout the

48 All

0003-9861/91 $3.00 Copyright 0 1991 by Academic Press, Inc. rights of reproduction in any form reserved.

GLUTATHIONE

REDUCTASE

cell. Experimentally, total cellular levels of GSSG are approximately 3000-fold higher than the level of GSSG expected from the equilibrium position of glutathione reductase (11). Changes in the redox status of the cellular glutathione redox buffer may be linked to changes in the redox state of specific protein sulfhydryl groups by thiol/disulfide exchange reactions, providing a potentially useful signal for control of biological activity in response to alterations in the cellular thiol/disulfide redox state (2, 3, 12, 13) or, alternatively, providing a rationalization for the often toxic effects associated with increased oxidation of the cellular glutathione pool (14, 15). The cellular glutathione redox status which would be attained at equilibrium of the glutathione reductase reaction approaches that required to maintain even the most difficult to reduce protein-SS-protein disulfides in their reduced form (12). Consequently, equilibrium of the glutathione reductase reaction would essentially preclude metabolic regulation by reversible thiol/disulfide exchange processes. Glutathione reductase must operate in the direction of GSSG reduction even in the presence of a vast excess of the product of the reaction, GSH. Inhibition of glutathione reductase by the approximately 10 mM intracellular GSH would significantly reduce the activity of glutathione reductase. For the yeast and erythrocyte enzymes, Mannervik (16) observed that GSH is an inhibitor of glutathione reductase; however, inhibition constants were not reported. In addition, no information is available on the behavior of rat-liver glutathione reductase with respect to inhibition by GSH. Since this organ has provided much of the experimental data on metabolic changes in GSH and GSSG levels, the inhibition of rat-liver glutathione disulfide reductase by GSH was studied in quantitative detail. The inhibition of rat-liver glutathione reductase by GSH is complex, depending on the square of the GSH concentration. The results suggest a sensitive and finetuned mechanism for steady-stat,e regulation of the glutathione redox status of cells within threefold of that predicted from measurements of total cellular GSSG. MATERIALS

AND

METHODS

Materials. Glutathione, glutathione disulfide, NADPH, CM-cellulose, and Z’,5’-ADP-Sepharose were purchased from Sigma. Dithiothreitol was obtained from Boehringer Mannheim. Glass-distilled, deionized water was employed in all experiments. All other reagents were analytical reagent grade or better. All GSH, NADPH, and DTT solutions were prepared daily. Rat-liver glutathione reductase was purified, with some modifications, by the method of Carlberg and Mannervik (17). All gelfiltration steps were replaced by dialysis against the appropriate buffers, and chromatography on CM-cellulose was as described by Carlberg and Mannervik. Only a single chromatography on 2’,5’-ADP-Sepharose was performed. After eluting the affinity column with 0.5 M potassium phosphate, pH 7.5, the glutathione reductase was eluted with a linear gradient (O-O.5 mM) of NADPH in 0.05 mM potassium phosphate, pH 7.5. The specific activity of glutathione reductase was 210 /.mrol/min/mg protein when assayed at 1 mM GSSG, 0.1 mM NADPH in 0.2 M potassium

49

INHIBITION

phosphate buffer, pH 7.0, containing 2 mM EDTA, at 25.O”C. Protein concentration was determined by the method of Bradford (18). Under similar conditions, Carlberg and Mannervik (17) reported a specific activity of 270 pmol/min/mg protein at 30°C. Velocity measurements. The initial velocity of the glutathione reductase reaction was determined at 25.O”C in 0.2 M potassium phosphate buffer containing 2 mM EDTA and the indicated concentrations of GSH, GSSG, and NADPH. GSH concentrations were determined by the method of Ellman (Q = 13.6 X lo3 Me1 cm-‘) (19). GSSG concentrations were determined by reduction with excess NADPH in the presence of glutathione reductase. NADPH concentrations were determined by the absorbance at 340 nm (t340 = 6.23 X lo3 M-’ cm-‘). Absorbance and velocity measurements were made on a Beckman DU7 or a Varian DMS 200 spectrophotometer with a thermostatted cell compartment maintained at 25.O”C. To maintain NADPH concentrations at initial levels and to minimize the formation of NADP’, contaminating GSSG (l-2%) in solutions of GSH was removed by reduction with 10 mM DTT for 1 h before use. To minimize nonenzymatic reduction of GSSG by the DTT carried over into the assay, cells were temperature equilibrated in the absence of GSH, the GSH was added, and the reaction initiated immediately by the addition of enzyme. From the highest DTT concentration used (0.75 mM) and the second-order rate constant for reaction of DTT with GSSG (14 mM-’ min-‘) (ZO), less than 2% of the GSSG would be consumed by nonenzymatic reaction during a 2.min assay. This estimate was confirmed by experimental measurement. DTT, at the maximum level (0.75 mM) carried over into the assay, produced no significant change in absorbance (

Inhibition of glutathione disulfide reductase by glutathione.

Rat-liver glutathione disulfide reductase is significantly inhibited by physiological concentrations of the product, glutathione. GSH is a noncompetit...
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