The effects of glutathione on protein thiols and α-tocopherol in rat liver microsomes following storage and during NADPH-dependent lipid peroxidation

Nutrition Research, Vol. 15. No. 8, pp. 1159-l 172, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 027 I-53 17/95 $9.50 + .oO

Pergamon

THE EFFECTS OF GLUTATHIONE ON PROTEIN THIOLS AND wTOCOPHEROL IN RAT LIVER MICROSOMES FOLLOWING STORAGE AND DURING NADPHDEPENDENT LIPID PEROXIDATION R.W. Scholz’*b, Ph.D.,

M.R. Yudt”, B.S., A.K. Saini”, Ph.D. and C.C. Reddyasb, Ph.D.

“Department of Veterinary Science and bEnvironmental Resources Research Institute, Pennsylvania State University, University Park, PA 16802 USA

ABSTRACT Liver microsomes from rats fed a diet supplemented with vitamin E (+E) were stored at 2°C in Tris buffers containing 5.0 mM reduced glutathione (GSH), GSH + 2.5 mM glutathione disulfide (GSSG) or no additions for up to 4 days. Thoroughly washed microsomes were then subjected to NADPH-dependent lipid peroxidation assays containing glutathione in combinations of its reduced and oxidized forms. These experiments demonstrated the presence of at least two glutathione-dependent factors in the inhibition of microsomal lipid peroxidation in rat liver. The first was quite labile and required GSH for its expression. The second factor was comparatively stable and showed a marked potentiation of GSH by GSSG. Changes in membrane protein thiols (PrSH) and cr-tocopherol (IX-TH) were also determined during storage of the microsomes in the various buffers and during assays of lipid peroxidation. Total membrane PrSH was higher in native (freshly prepared) liver microsomes from +E rats than in microsomes from rats fed a diet deficient in vitamin E (-E). The addition of GSH or GSH + GSSG diminished the extent of PrSH depletion during storage in both +E and -E microsomes, but the protective effect was greater for the +E microsomes. The loss of (r-TH was also reduced in microsomes stored in buffers containing either GSH or GSH + GSSG compared to Tris buffer alone. Changes in PrSH during lipid peroxidation were pronounced in the absence of GSH or GSH + GSSG and were independent of the ar-‘III content of the membranes. Addition of either GSH or GSH + GSSG to the assays markedly reduced the depletion of PrSH during lipid peroxidation. For +E microsomes, the depletion of PrSH and (r-TH in the presence or absence of GSH were parallel. The rate and extent of this depletion, however, were much greater in the absence of GSH. These data demonstrated that membrane (r-TH was not essential in maintaining PrSH in the absence of GSH once lipid peroxidation was initiated. It also appeared that the potentiating effect of GSH by GSSG on the inhibition of rat liver microsomal lipid peroxidation, which was also resistant to inactivation during storage, was not through selective maintenance of a-TH or general sparing of PrSH. KEY WORDS:

Glutathione,

Protein thiols, cY-Tocopherol, Lipid peroxidation

Corresponding author: R.W. Scholz, Department of Veterinary Science, 122 AS1 Building, Pennsylvania State University, University Park, PA 16802 USA 1159

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INTRODUCTION

Reduced glutathione (GSH) is the most abundant non-protein thiol found in mammalian cells and in rat liver its concentration normally may exceed 5 mM (1). It is also well established that depletion of cellular GSH leads to a number of adverse effects, including reduced or compromised detoxification of xenobiotics, depletion of protein thiols (PrSH), enhanced peroxidation of membrane lipids, among others (2). Previous investigations reviewed in (3) have demonstrated the presence in rat liver of GSH-dependent cytosolic and membrane factors that inhibit peroxidation of microsomal membrane lipids. In some of these reports, a dependency specifically on membrane-associated cw-tocopherol (wTH) for the GSH effect has been claimed (4-8) whereas in others it has not (9-11). In experiments where dependency on r~-TH was observed, it was proposed that GSH functioned with a heat labile microsomal factor in recycling (Y-TH from the a-tocopheroxyl radical ((Y-T*)(4,5). This proposal has been supported by McCay, et al. (6) where it was suggested that wTH functions as an electron shuttle for a “free radical reductase” wherein a labile factor, ostensibly a catalytic protein, transfers a hydrogen atom from GSH to regenerate (r-TH from a-T.. Similar observations have been reported by Packer, et al. (12) where GSH was shown to be effective in the presence of NADPH in reducing the occurrence of the chromanoxyl radical in microsomal and mitochondrial membranes. More recently, RobeyBond, et al. (13) have indicated that GSH enzymatically regenerates wTH from WT. in rat liver microsomes. Isolation and characterization of this putative protein factor has remained elusive, however. In addition to the GSH-dependent factor, preliminary experiments from our laboratory have indicated an additional rat liver microsomal factor that inhibits lipid peroxidation in the presence of GSH and its oxidized form, glutathione disulfide (GSSG) (14). This second factor, in which a potentiation of GSH by GSSG was shown, appeared to be independent of microsomal wTH because some protection against lipid peroxidation was observed in liver microsomes from rats severely depleted of wTH by dietary means. The present experiments were designed to provide additional evidence for the glutathione-dependent factors in rat liver microsomes that inhibit peroxidation of membrane lipids. These studies also examined changes in membrane PrSH and wTH in peroxidizing microsomes and in microsomes stored for up to four days in several buffers varying in GSH/GSSG content.

EXPERMENTAL Chemicals The NADPH, GSH, GSSG, glutathione reductase, bovine serum albumin, butylated hydroxytolune, trichloracetic acid, thiobarbituric acid, guanidine*HCl, and 5,5’-dithiobis (2nitrobenzoic acid) (DTNB) were obtained from Sigma Chemical Company (St. Louis, MO). CYTocopherol, y-tocopherol, and tocopherol-stripped corn oil and lard were purchased from Eastman Kodak Company (Rochester, NY). Ingredients for preparation of the experimental diets to include torula yeast, DL-methionine, mineral mixture (USP XXI), and the fat and water soluble vitamins were products of United States Biochemical Corporation (Cleveland, OH). All other chemicals were of reagent grade quality.

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Animals and diets Male Long-Evans hooded rats, obtained at weaning (Charles River Laboratories, Wilmington, MA) were fed chemically-defined, torula yeast-based diets containing tocopherolstripped corn oil and lard as fat sources. The composition of the control diet, which is deficient in vitamin E, selenium and chromium is presented in Table 1. A second diet was supplemented with vitamin E as RRR-a-tocopherol acetate to contain 150 III/kg of diet. Both diets were supplemented with selenium as sodium selenite (0.5 mg/kg) and chromium as chromium chloride hexahydrate (0.2 mg/kg). Animals were fed their respective diets for 10 weeks.

TABLE 1 Composition

of Control Diet

Ingredient Sucrose Torula yeast Tocopherol-stripped corn oil Tocopherol-stripped lard Cellulose Mineral mixture (USP XXI) Vitamin mixture (minus vitamin E)* DL-methionine Choline chloride (70%) *

-& 444.9 300.0 100.0 60.0 40.0 40.0 10.0 3.0 2.1

The vitamin mixture provided the following components in mg/kg of diet with sucrose serving as the carrier component: thiamine~HC1, 10.0; riboflavin, 10.0; nicotinic acid, 25.0; Ca pantothenate, 20.0; pyridoxine*HCl, 10.0; biotin, 0.10; folic acid, 0.20; inositol, 100.0; vitamin B,, (0.1% mix), 20.0; ascorbic acid, 50.0; vitamin A (retinyl acetate), 6.3; vitamin D-3 (crystalline), 0.075; and menadione sodium bisulfite (trihydrate), 0.50.

Preparation

of liver microsomes

The rats were anesthetized with Na pentobarbital, 30 mg/kg body wt. i.p., and exsanguinated by severing major blood vessels upon opening of the thoracic cavity. Livers were removed, rinsed with 0.87% NaCl, and homogenized on ice in a motor-driven Potter-Elvehjem homogenizer with 9 vol (w/v) of 0.25 M sucrose buffered to pH 7.4 with 10 mM Tris. The crude homogenate was centrifuged at 2-4°C at 12,000 xg for 20 min and the resulting supematant centrifuged again under identical conditions. Microsomes were sedimented from the 12,000 xg supematant by centrifugation at 105,000 xg for 1 hr at 2°C. The microsomal pellet was resuspended in 0.15 M Tris buffer, pH 7.4, and centrifuged again at 105,000 xg for 1 hr at 2°C. This washing procedure was repeated one additional time. Microsomes were concentrated in 0.15 M Tris buffer, pH 7.4, to provide a protein concentration of 5-7 mg/ml. For experiments on the effects of storage conditions on lipid peroxidation, PrSH and (Y-TH, liver microsomes were uniformly suspended in one of three buffers: a) 0.15 M Tris, pH 7.4; b) Tris buffer supplemented with 5 mM GSH; and c) Tris buffer supplemented with 5 mM GSH + 2.5 mM GSSG. Microsomal suspensions were stored refrigerated in each of the buffers at 2°C for up to 4 days. Prior to assays of lipid peroxidation, PrSH and (r-TH, microsomes were sedimented by centrifugation at 105,000 xg for 1 hr at 2°C. Microsomes were washed in Tris

R.W. SCHOLZ et al.

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buffer and sedimented as described above. This process was repeated an additional time and the final wash revealed lack of contaminating GSH or GSSG as determined by the methods of Tietze (15). Lipid peroxidation

assay

Lipid peroxidation was assessed by the formation of thiobarbituric acid (TBA)-reactive products in a NADPH-dependent enzymatic system containing 12 PM FeSO,, 0.25 mM NADPH, 0.05 M Tris buffer, pH 7.4, and varying concentrations of GSH and GSSG which were dissolved in distilled water and the pH adjusted to 7.4 prior to their addition to the assays (16). Approximately 0.2 mg of microsomal protein/ml of reaction mixture was used in these experiments. Assays were conducted over a time-course of 2 hours and aliquots removed at various intervals for the quantitation of TBA-reactive products at 535 nm using an extinction value of 1.56 x 105*M‘1cm-’ (17). Determination

of cu-tocopherol

Aliquots of freshly prepared or stored microsomes containing approximately 1 mg of microsomal protein were prepared for assay of wTH as described previously (16). cu-Tocopherol was assayed by a reversed phase HPLC procedure modified after Bieri, et al. (18). Separation was accomplished on a 250 mm x 4.6 mm ODS column, 5 pm particle size, fitted with a 50 mm x 4.6 mm ODS precolumn (Supelco, Inc., Bellefonte, PA) using isocratic elution, 2.4 ml/min, with a mobile phase of 97.1% methanol and 2.9% distilled water. Quantitation of (r-TH was with UV (290 nm) or fluorescence (295 nm excitation; 330 nm emission) detection using a known amount of y-TH as internal standard and a standard curve containing known amounts of wTH. Protein sulfhydryl

assay

Protein thiols were determined by the method of DiMonte, et al. (19), with modifications (20,21). Microsomal preparations containing approximately 4 mg protein/ml were precipitated with TCA at a final concentration of 6.5% and the precipitate sedimented by centrifugation at 3,000 xg for 10 min. Following two washes with TCA, the protein pellet was washed an additional time with 5 ml of methanol:diethyl ether (1:5 v/v), sedimented by centrifugation and dried. The dried pellet was dissolved in 1 ml of 6 M guanidine.HCl to which was added 1 ml of 0.625 M Tris, pH 7.6, with vigorous mixing on a vortex mixer. Fifty ~1 of 5 mM DTNB was added, the solution mixed, and the absorbance determined at 412 nm after 20 min. The data were expressed as nmoles SH per mg of protein based on a GSH calibration curve. Statistics All data were subjected to analysis of variance, general linear models procedure (Minitab@ Statistical Software, 1991 version, State College, PA). Time-course measurements were subjected to repeated measures analysis of variance. Significant differences (P < 0.05) among treatment means were determined by the Student-Newman-Keuls multiple range procedure (22). Lag phase durations involving lipid peroxidation data were estimated by extrapolating the rapidly rising linear portion of TBARS to the baseline level (7).

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RESULTS

Results of experiments with native (freshly prepared) microsomes from rats fed the vitamin E supplemented diet, and the effects of GSH, GSSG, and GSH + GSSG on in vim lipid peroxidation, are presented in Fig. 1A. Lipid peroxidation was monitored over a time-course of 2 hours and is expressed as nmoles TBARS formed/mg microsomal protein. Addition of 5 mM GSH to the assay system produced the well established lag in formation of TBARS. The addition of GSH + GSSG, (5.0 and 2.5 mM, respectively) greatly enhanced the lag period. The addition of 2.5 mM GSSG alone was without effect on inhibition of lipid peroxidation under any of the assay conditions employed, as shown in Fig. lA, and these data are removed from the remaining panels for clarity. Additional assays were conducted with microsomes stored in 150 mM Tris buffer, pH 7.4, or buffer + 5 mM GSH or buffer + 5 mM GSH + 2.5 mM GSSG for 2 days (panels B,C and D) or 4 days (panels E,F and G). Prior to the lipid peroxidation assays microsomes were sedimented by centrifugation and washed 2 times with buffer. No GSH or GSSG could be detected in the supematant from the final washing. The data show that the GSHdependent protection against lipid peroxidation was lost prior to 2 days in microsomes stored only in Tris buffer. The GSH + GSSG effect, however, was still present (approximately 40% that of fresh microsomes) after storage in Tris buffer alone for 48 hours (compare panel B with panel A). This effect was removed completely within 4 days storage in Tris buffer alone (panel E). Storage of microsomes in buffers containing GSH or GSH + GSSG for 2 days (panels C and D) or 4 days (panels F and G) produced only modest reductions in the inhibition of lipid peroxidation compared with fresh microsomes (panel A). Collectively the data in Fig. 1 demonstrate the presence of at least two GSH-dependent factors that inhibit microsomal lipid peroxidation. The first was quite labile and required GSH for its expression; the second was comparatively stable and required GSH + GSSG for its expression (GSSG alone was without effect). Heat treatment of microsomes at 100°C for 10 min completely removed the potentiation of GSH by GSSG using a nonenzymatic ascorbate/Fe3+/ADP system to initiate lipid peroxidation (data not shown). Changes in membrane PrSH during four days’ storage in each of three different buffers are presented in Fig. 2. In these experiments, we additionally compared microsomes from vitamin E supplemented (+E, panel A) and vitamin E depleted (-E, panel B) rats. For freshly prepared +E microsomes, the (w-TH concentration was 0.68 + 0.05 nmoles/mg protein whereas for -E microsomes this value was 0.05 f 0.005 nmoleslmg protein (mean f. SEM, n = 6). The data show a significant (PCO.05) depletion of PrSH over time in microsomes stored in Tris buffer alone and this effect was greater in membranes depleted of cr-TH. Addition of either GSH or GSH + GSSG to the Tris buffer diminished the extent of PrSH depletion in +E microsomes and there were no significant (P>O.O5) differences between either system for this effect, In -E microsomes, however, the effect of either GSH or GSH + GSSG on attenuating the loss of PrSH during storage was removed. Additionally, the inclusion of GSH + GSSG in the Tris buffer resulted in a greater loss in PrSH compared to the inclustion of GSH alone. The addition of buffered 2.5 mM GSSG alone to the microsomes produced changes during storage similar to that for microsomes stored only in Tris buffer (data not shown).

R.W. SCHOLZ et al.

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1. Effects of assay and storage conditions on NADPH/Fe’+-dependent lipid peroxidation in liver microsomes from rats fed a diet supplemented with vitamin E. Each point is a mean value from 6 animals. Approximately 0.2 mg of microsomal protein/ml of reaction mixture was included in the assays. The initial concentrations of GSH and GSSG were 5.0 mM and 2.5 mM, respectively. Error bars are not included in the figures for the purpose of clarity. Pooled standard error of the means for panels A-G are 5.4, 4.3, 3.5, 3.4, 4.6, 4.4 and 4.4, respectively. An asterisk (*) indicates significant difference (P
MICROSOMAL LIPID PEROXIDATION

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Changes in membrane protein thiol (P&H) content in stored microsomes from +E (panel A) and -E (panel B) rats. Each bar + SEM represents a mean value from 6 animals. Microsomes were stored in each of three buffers at 2°C for up to four days. Means with different lettered overscripts within each buffer are statistically different (P < 0.05).

The changes in ar-TH concentration of rat liver microsomes as affected by storage buffer and time of storage are shown in Fig. 3. In this experiment, microsomes were prepared from rats fed the diet supplemented with vitamin E and the IX-TH content of the freshly prepared microsomes prior to storage, day 0, was 0.68 _+ 0.05 nmoles/mg protein (mean f SEM, n = 6). The data show a statistically significant (P
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Changes in wtocopherol concentration of microsomal membranes stored in each of three buffers at 2°C for up to four days. Microsomes were prepared from rats fed the diet supplemented with vitamin E. See legend for Fig. 2 for additional details.

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We examined the changes in PrSH in rat liver microsomes undergoing NADPH/Fe*+dependent lipid peroxidation. It was our interest to monitor these changes in microsomes from rats fed diets either supplemented (+E) or deficient (-E) in vitamin E as well as the effects of GSH or GSH + GSSG. These data are presented in Fig. 4. In these experiments the microsomal (r-TH concentration was 0.65 f 0.06 and 0.07 f 0.005 nmoles/mg protein (mean f SEM, n = 6) for +E and -E rats, respectively. In the control assay system without GSH, PrSH depletion was pronounced and represented a loss of approximately 60% within 30 minutes, independent of the CY-THcontent of the microsomes. A similar accelerated rate of PrSH loss was also observed in the assay system containing 2.5 mM GSSG. Addition of GSH or GSH + GSSG to the assays markedly reduced the depletion of PrSH and there were no apparent differences between either system for this effect. Interestingly, the rates of PrSH depletion during the time-course assays were essentially identical for +E and -E microsomes, although statistical analysis of the combined data showed the initial PrSH concentration prior to initiation of lipid peroxidation to be significantly higher (P
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Changes in membrane protein thiol (PrSH) content in peroxidizing micrasomes from +E (panel A) and -E (panel B) rats. Standard error of the means for control, GSH, GSSG and GSH + GSSG assay systems in panel A were 2.2, 2.2, 3.6 and 2.4, respectively. Corresponding values for the data in panel B were 2.3, 1.2, 2.4 and 1.2, respectively. See legend for Fig. 1 for additional details.

Changes in (r-TH and PrSH of peroxidizing microsomes prepared from +E rats in the presence (panel A) or absence (panel B) of 5 mM GSH are shown in Figure 5. Data are presented as percentage change from zero time, or the time of initiation of lipid peroxidation.

MICROSOMAL LIPID PEROXIDATION

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Initial concentrations of o-TH and PrSH were 0.61 + 0.05 nmoles/mg protein and 53.3 f 4.2 nmoles/mg protein, respectively (mean + SEM, n = 6). In either the presence or absence of GSH, losses of CY-THand PrSH during lipid peroxidation were essentially parallel, although the rate and extent of depletion of both constituents was greater in the absence of GSH. Statistical analysis of the combined data in Fig. 5 revealed that microsomal concentrations of (r-TH and PrSH in the absence of GSH were all significantly lower (P< 0.05) than in the presence of GSH, beginning at the 10 min interval after initiation of lipid peroxidation. Results essentially identical to that illustrated in Flg. 5A were obtained when GSH + GSSG were substituted for GSH alone (data not shown).

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Percentage change in cu-tocopherol and protein thiols in peroxidiiing microsomes in the presence (panel A) and absence (panel B) of 5 mM GSH. Pooled standard error of the means for (w-TH and PrSH in panel A were 2.3 and 2.2, respectively. Corresponding values for the data in panel B were 4.4 and 4.0 respectively. See legend for Fig. 1 for additional details.

DISCUSSION

The presence of at least two microsomal factors in rat liver that inhibit lipid peroxidation, using a NADPH/Fe’+ system to stimulate the process, was clearly shown by aging membranes in Tris buffers containing GSH, GSH + GSSG or buffer alone. One of these factors, dependent on GSH for its effect, was comparatively unstable and protection against lipid peroxidation was lost completely within 48 hours when stored in Tris buffer alone. Under identical storage conditions in vitro an additional inhibitory effect, dependent on GSH + GSSG, was still active. When GSH or GSH + GSSG were included in the storage buffers, this factor was remarkably

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R.W. SCHOLZ et al.

stable and afforded protection against lipid peroxidation in microsomes aged for at least 96 hours. The protective effects of GSH or GSH + GSSG in these experiments may be related to a number of factors, including preservation of membrane (r-TH, maintenance of protein thiols, among others. Protein sulfhydryls, in addition to a-TH, are proposed to protect biomembranes from oxidant attack. In this context it is thought that membrane thiols compete with a-TH for trapping radicals, thus sparing (w-TH under oxidizing conditions (23). It has also been shown that loss of PrSH can be prevented by GSH which has led some investigators to suggest that a GSH-dependent enzyme maintains PrSH in the reduced state under oxidizing conditions (11,24). Studies on changes in PrSH and CX-THduring peroxidation of microsomal membranes have been reported previously (11,21,25-27). In general, it is established that oxidative stress results in loss of both membrane PrSH and (Y-TH and GSH protects from this loss. However, the type of prooxidant used to initiate lipid peroxidation in vitro dramatically influences these interrelationships. In a lipid peroxidation system initiated by Fe”, for example, CX-THloss is rapid and coincides with the onset of lipid peroxidation (27). However, GSH was not included in this system which is an important consideration because GSH can markedly reduce the depletion of microsomal (r-TH and extend the lag period in a system using Fe*’ to initiate lipid peroxidation. It is therefore important to stress that (Y-TH is an effective inhibitor of lipid peroxidation in systems containing Fe’+ providing GSH is also present and that temporal relationships among various membrane constituents during oxidant stress are affected considerably by the in vitro system designed to study them. Alternatively, the maintenance of PrSH by GSH may be associated with the GSHdependent recycling of a-TH from reversible oxidation products. Our data have shown a loss of PrSH during the course of lipid peroxidation; however, we are not convinced this loss was totally dependent upon the presence of or-TH in the membranes because a similar rate of PrSH depletion was observed in microsomal membranes essentially devoid of a-TH. These data cast some doubt on an absolute requirement for (r-TH in maintaining PrSH in the reduced state in liver microsomes once lipid peroxidation is underway. It is noteworthy, however, that the total PrSH content was higher in microsomal membranes from +E rats, compared with membranes depleted of or-TH by dietary means. It may be speculated that critical membrane proteins are adversely affected in vitamin E deficiency and that whereas the rates of PrSH depletion during lipid peroxidation were similar for +E and -E microsomes (Fig. 4), the experimental procedures employed do not identify which specific protein thiols are lost during oxidative stress. Recently it has been proposed that the GSH-dependent protection against lipid peroxidation in rat liver microsomes involves the maintenance of PrSH (11,21), and hence ol-TH (26), in the reduced state. Additionally, a dependency specifically for a-TH was not shown to be required for the inhibitory effect because other tocopherol isomers were also able to protect against lipid peroxidation (11). In isolated hepatocytes, however, others have shown that vitamin E prevents PrSH modification, even in cells that are severely depleted of GSH (28,29). Protein thiol depletion in the storage experiments (Fig. 2) was considerably greater in -E microsomes than in +E microsomes. Under these experimental conditions, where the presence of membrane CXtocopherol attenuated PrSH loss, GSH further enhanced this effect. The differences observed in PrSH loss during storage as compared with time-course assays of lipid peroxidation may be explained, in part, by the stronger oxidizing conditions of the latter. The GSH-dependent lipid peroxidation inhibitory factor in rat liver microsomes, which is both heat labile and sensitive to trypsin treatment (4,6,9,10,21), has been proposed to act as a “free radical reductase” wherein cr-TH is reversibly recycled from a-T* during oxidative stress (6). This protection has also been proposed to be associated with microsomal glutathione S-transferase (GST) (30-32) as well as with membrane associated phospholipid hydroperoxide glutathione peroxidase activities (33). Other

MICROSOMAL LIPID PEROXIDATION

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investigations, however, have not attributed the GSH-dependent inhibitory effect on rat liver microsomal lipid peroxidation on these enzymes (34,35). Thus, interactions among (Y-TH, PrSH, GSH, and various glutathione-dependent enzymes in inhibition of lipid peroxidation in isolated rat liver hepatocytes or microsomes appear complex and precise mechanisms associated with these interactions remain to be established. A second factor, dependent upon the presence of both GSH and GSSG, is considerably more resistant to inactivation during storage at 2°C than its GSH-dependent counterpart. The mechanism associated with the inhibitory effect of GSH + GSSG on lipid peroxidation has not been established. Previous work in our laboratories has shown that inhibition by this heat labile factor is not dependent upon microsomal cr-tocopherol (14). Reduction of lipid hydroperoxides to their corresponding alcohols by a protein activated by GSSG through thiol/disulfide exchange may be involved. The oxidation of PrSH by disulfides, or S-thiolation, results in the formation of protein mixed disulfides (36). This exchange reaction has been demonstrated to affect the activity of a number of enzymes, either as up or down regulation, providing the reaction is reversible. Indeed, protein mixed disulfide formation has been proposed as a mechanism to protect SH groups of proteins from inactivation, for example, during oxidative stress (37). We have previously suggested this as an explanation for our observations on enhanced protection against lipid peroxidation by GSH + GSSG (38). Whether S-thiolation is importantly involved in the GSH + GSSG protection is arguable, however. Although we did observe an attenuation of PrSH loss by GSSG alone during lipid peroxidation in +E microsomes, it is emphasized that precise identification of specific (or critical) protein thiols that may be affected was not possible with the experimental procedures employed. Any potential involvement of reversible S-thiolation reactions, particularly in relationship to physiologically important thiol/disulfide redox pairs, and their effects on membrane lipid peroxidation, remain to be determined.

ACKNOWLEDGEMENTS

The authors thank G.C. Loop and L.J. Muchinsky supported in part by grant HL 31245 from NIH.

for their assistance.

This work was

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Accepted

for

publication

March

6, 1995.

of enzyme thiols-disulfides

in metabolic