Pyruvate Kinase Is Protected by Glutathione-Dependent Redox Balance in Human Red Blood Cells Exposed to Reactive Oxygen Species

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1 October 2008 Biol. Pharm. Bull. 31(10) (2008) 1875 Pyruvate Kinase Is Protected by Glutathione-Dependent Redox Balance in Human Red Blood Cells Exposed to Reactive Oxygen Species Yuki OGASAWARA,* Masayo FUNAKOSHI, and Kazuyuki ISHII Department of Environmental Biology, Meiji Pharmaceutical University; Noshio, Kiyose, Tokyo , Japan. Received May 20, 2008; accepted August 4, 2008 To determine the antioxidant role of glutathione (GSH) in human red blood cells (RBCs), we investigated the effect of disrupting GSH homeostasis on the oxidative modification of thiol-dependent enzymes by exposure to tert-butyl hydroperoxide (BHP). When hemolysate was incubated with BHP, significant decreases in enzyme activity were observed. However, the inactivation did not occur in intact RBC suspensions that were exposed to BHP. In this study, we used two independent treatments aimed at decreasing the level of reduced form of GSH, pre-incubation with a glutathione reductase inhibitor or glucose-free medium to examine the influences of preventing GSH-dependent antioxidant and reactivation activity on thiol-dependent enzyme. Pyruvate kinase (PK) activity clearly decreased along with depletion of GSH compared to other glycolytic enzyme activities by BHP exposure in RBCs. The addition of GSH, but not glucose, before BHP exposure completely prevented the inactivation of PK in hemolysate; however, partial reactivation of inactivated PK was observed by post-addition of both GSH and glutaredoxin at an early stage during BHP exposure. Moreover, hydroxyl radicals but not hydrogen peroxide inactivated PK. These results suggest that PK is highly susceptible to radicals and that GSH is essential to protect PK activity by not only directly scavenging radicals but also by systematically reactivating oxidized enzyme in human RBCs. Key words oxygen species oxidative stress; pyruvate kinase; human red blood cell; protein modification; tert-butyl hydroperoxide; reactive Proteins are susceptible to attack in the presence of reactive oxygen species (ROS). Several types of protein damage have been identified, including amino acid modification, cross-linking, and degradation. 1,2) Sulfhydryl residues are among the most easily oxidized protein residues, and oxidation can lead to intermolecular cross-linking of proteins as well as enzyme inactivation. 3) Red blood cells (RBCs) have been used as a model system in numerous investigations to determine the significance of the glutathione-dependent redox system 4,5) because RBCs have an efficient antioxidant defense system that prevents oxidative damage. 6 8) Although most cells are susceptible to oxidative injury, RBCs are particularly at risk due to their high iron content and regular exposure to high concentrations of oxygen, as well as environmental chemicals and drugs. tert-butyl hydroperoxide (BHP) is a commonly used compound to investigate the effects of peroxides and radicals on biological systems. 6 9) Many lines of evidence have emerged to support a fundamental role of redox modification of certain proteins in normal cellular biochemical and physiological processes. A number of purified proteins are sensitive to oxidation by thiol disulfide interchange reactions. These include enzymes involved in both central and peripheral metabolism, such as glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as well as creatine kinase, aldehyde dehydrogenase, and carbonic anhydrase. 10,11) In particular, glycolytic and related enzymes are susceptible to oxidation through the formation of mixed disulfides with low molecular weight thiols. 12) Thus, reduced form of glutathione (GSH) is likely to contribute to the protection of all thiol-dependent enzymes against ROS in the cell. However, real susceptibility of the enzymes to ROS and the protective effects of GSH against oxidative stress in human RBCs remains poorly understood. In this study, we focused on the reactivity of thiol-dependent enzymes related to glucose metabolism in human RBCs upon exposure to BHP. To elucidate a physiological antioxidant role for GSH and real ROS-susceptible proteins in RBCs, two independent treatments were designed to deplete GSH. First, pre-treatment with an inhibitor of glutathione reductase (GR) following exposure to BHP decreased GSH levels in RBCs without recovery of the levels. Second, RBCs were pre-incubated in glucose-free medium following exposure to BHP to significantly decrease GSH levels, which did not recover well. When GSH levels failed to recover after BHP exposure, we found that pyruvate kinase (PK) activity was significantly decreased following exposure to BHP in intact RBC. To estimate whether direct or systematic effects of GSH contribute to protect PK activity by BHP exposure, we observed the alteration of PK activity in a reconstituted system with hemolysate. Furthermore, the specificity of the PK inactivation was investigated in detailed experiments using purified PK and ROS. MATERIALS AND METHODS Chemicals D-Fructose 6-phosphate, phosphoenolpyruvate, reduced glutathione (GSH), D-fructose 1,6-diphosphate, BHP, glutaredoxin (Grx) from Escherichia coli, aldolase from rabbit muscle, glycerol-1-phosphate dehydrogenase, and a triosephosphate isomerase mixture from rabbit muscle, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and oxidized glutathione (GSSG) were purchased from Sigma Chemical Co. Dithiothreitol (DTT), dithiobis-2-nitrobenzoic acid (DTNB), ascorbic acid, hydrogen peroxide (atomic absorption analysis grade), NADPH, NADP, NADH, ADP, ATP, G6PDH from yeast, and glucose assay kits were obtained from Wako Pure Chemical Co. Lactate dehydrogenase (LDH) from pig heart and bovine serum albumin were obtained from Boehringer-Mannheim-Japan Co. All other To whom correspondence should be addressed. yo@my-pharm.ac.jp 2008 Pharmaceutical Society of Japan

2 1876 Vol. 31, No. 10 reagents were of the highest grade commercially available. Preparation of Human Red Blood Cell Suspensions and Hemolysate Samples Blood samples were obtained from eight healthy subjects (aged 22 43, 12 h-fasting) with informed consent. Blood was drawn into Vacutainer tubes containing ethylenediamine tetraacetic acid (EDTA). RBCs were prepared by centrifugation for 15 min at 4 C and 800 g and were washed twice in phosphate buffered saline (PBS) with the same centrifugation procedures. RBC suspensions were prepared by adding PBS in the presence or absence of 10 mm glucose. The hematocrit of each RBC suspension was adjusted to 40% (Hct. 0.40). Hemolysates were prepared by adding and mixing eight volumes of distilled water to washed RBCs, which were cooled after the addition of one volume of 10-fold concentrated PBS. Finally, the hemolysate was prepared in a PBS matrix as a ten-fold dilution of packed RBCs. All experiments were performed on the day of the blood draw. Exposure of Hemolysate and RBC Suspensions to BHP The effects of BHP on hemolysate were examined following exposure to 0.5 mm BHP. After a 60 min incubation at 37 C, 10 or 20 ml of hemolysate was removed to evaluate the residual activity of various enzymes. In addition, a RBC suspension containing 10 mm glucose was incubated at 37 C with 2.0 mm BHP for 60 min, after which the RBCs were washed twice to remove extracellular BHP. The RBC pellet obtained was lysed by the addition of eight volumes of sterilized distilled water, which was followed by the addition of one volume of 10-fold concentrated PBS to condition the lysate. Disruption of GSH Homeostasis RBC suspensions (Hct. 0.40) were pre-incubated at 37 C in the presence or absence of 1 mm BCNU in PBS containing 10 mm glucose. After a 30 min incubation, each suspension was washed twice with PBS containing glucose. Ten microliters of BHP was added to 0.49 ml of RBC suspension, followed by incubation at 37 C for various time. RBCs with low intracellular glucose levels (less than 2.0 mm in RBC) were prepared by pre-incubation for 45 min at 37 C in a PBS suspension in the absence of glucose. Each RBC suspension (Hct. 0.40) was incubated in the presence or absence of 2.0 mm BHP for various times. RBC glucose concentrations were determined by an enzymatic method using a glucose test kit. GSH and GSSG Determination RBCs in suspension (Hct. 0.40) were centrifuged, and the packed cells were lysed by the addition of eight volumes of distilled water and the immediate addition of one volume of 25% (w/v) sulfosalicylic acid. After centrifugation for 5 min at g, clear supernatant was used for determination of GSH and GSSG. GSH was measured by a HPLC method previously reported 13) with minor modifications. Briefly, the clear supernatant (10 ml) of acid-treated samples was neutralized by addition of four volume of 0.2 M borate buffer (ph 10.5), and the mixture (50 ml) was reacted with labeling reagent in the presence of tris(2-carboxyethyl)-phosphine (TCEP) to determine total GSH. The reduced form of GSH was measured without reduction step by TCEP, and GSSG value was calculated by subtracting the amount of reduced GSH from total GSH amounts. The concentration of GSH and GSSG in packed RBCs was calculated by multiplying the value in the lysate by ten, respectively. Assays of Human Erythrocyte Enzyme Activity PK activity was measured spectrophotometrically at 340 nm using LDH as a coupling enzyme. 14) The assay system contained in 1 ml at 37 C: 2 mm ADP, 2 mm phosphoenolpyruvate, 200 m M NADH, 65 mm KCl, 6.8 mm MgCl 2, 30 units of LDH, 50 mm triethanolamine (ph 7.4), and 10 ml of lysate to be assayed. Catalase activity was determined using the method of Cohen et al. 15) Superoxide disumutase (SOD) was assayed by the method of McCord and Fridovich. 16) Glutathione S-transferase (GST) activity was determined by the method of Strange et al. 17) Glutathione peroxidase (GPX) activity was determined using the method of Flohé et al. 18) and Grx activity was determined according to the method of Mieyal et al. 19) Other RBC enzymes were assayed according to methods of the International Committee for Standardization 20) with minor modifications, and all assays were carried out at 37 C in the absence of thiols (reducing agents). A unit of enzyme was defined as the amount catalyzing the conversion of 1 mmol of substrate per min under each assay conditions. Protective Effects of GSH and Glucose on PK Inactivation by BHP Exposure in Hemolysate To investigate the direct effect of GSH and glucose, PK activity was examined in hemolysate after exposure to 0.5 mm BHP. Following incubation for 60 min at 37 C in the presence of 2 mm GSH or 10 mm glucose, a suitable portion was taken to assay the residual enzyme activity. Reactivation of PK Activity in Reconstituted Hemolysate Using the GSH-Dependent Redox System To examine the reversibility of the enzyme modifications, the effects of GSH or the GSH-dependent redox system, including NADPH and Grx, on reactivation were observed. Hemolysates diluted with PBS were incubated with 0.25 mm BHP. After incubation at 37 C for 15 min, 5.0 mm GSH or 5.0 mm GSH plus 0.20 mm NADPH and 30 m M Grx were added to the hemolysate and incubated for an additional 30 min. A portion of each mixture was taken to assay of PK activity at various time intervals. Specific Inactivation of PK Following Exposure to Hydroxyl Radicals Purified rabbit muscle PK was exposed to hydroxyl radicals generated by incubating the following reagents at the indicated final concentrations in 0.5 ml of PBS (ph 7.4) at 37 C: 1) 1 mm H 2 O 2, 2) 20 m M FeCl 3, 100 m M EDTA, and 50 m M ascorbic acid, and 3) 1 mm H 2 O 2, 20 m M FeCl 3, 100 m M EDTA, and 50 m M ascorbic acid. After 60 min, residual activities were determined in all reaction mixtures. Protective Effects of GSH and Glucose on PK Inactivation by Hydroxyl Radical Exposure To elucidate the direct effect of GSH and glucose, purified rabbit muscle PK was exposed to hydroxyl radicals generated by incubating with 1 mm H 2 O 2, 20 m M FeCl 3, 100 m M EDTA, and 50 m M ascorbic acid. Following incubation for 60 min at 37 C in the presence of 2 mm GSH or 10 mm glucose, a suitable portion was taken to assay the residual enzyme activity. Other Methods Protein was assayed by the method of Lowry et al. 21) using bovine serum albumin as a standard (Bio-Rad Protein Assay Kit). Statistics Data are expressed by the mean S.D. and compared using Student s t test or analysis of variance (ANOVA). Results were considered statistically significant

3 October when p RESULTS Effect of BHP Exposure on Enzyme Activities within the Hemolysate and RBC Suspension To examine the influence of BHP exposure on the activities of various enzymes, including glycolytic, pentose phosphate pathway, and antioxidant enzymes, hemolysate was incubated at 37 C for 60 min in the presence of 0.5 mm BHP. As shown in Table 1, significant inactivation of hexokinase (HK), PFK, GST, GPX, GAPDH, G6PDH and PK was observed following incubation with BHP, while a significant decrease in GR, SOD, Grx, and catalase was not observed upon exposure of the hemolysate to BHP. When 2.0 mm BHP was added to an intact RBC suspension containing glucose at 37 C for 60 min, GST and GPX activities were significantly decreased compared to the control values, while the significant decreases seen in the activities of HK, PFK, GAPDH, G6PDH, PK disappeared (Table 2). Alterations in GSH Homeostasis and Enzymatic Activities upon Exposure of RBC to BHP In the presence of glucose, GSH and GSSG levels in intact RBCs transiently decreased and increased upon exposure to BHP but rapidly Table 1. Effects of BHP on Various Enzyme Activities in RBC Lysate Enzyme Remaining activity (%) a) Control BHP b) Glutathione reductase Glucose-6-phosphate dehydrogenase * Pyruvate kinase * Phosphofructokinase * Glutathione peroxidase * Glyceraldehyde-3-phosphate * dehydrogenase Hexokinase * Superoxide dismutase Catalase Glutathione-S-transferase * Glutaredoxin a) Activities were expressed % (mean S.D.) for the values with incubation in the absence of BHP. b) Incubation: 0.5 mm BHP for 60 min. p 0.05 (for control). recovered within 60 min (Figs. 1A, B). BCNU, an inhibitor of GR, 22) was used to deplete GSH in RBCs. After treatment with 1 mm BCNU and 10 mm glucose, the RBC suspension was exposed to BHP and incubated for 1 h in the presence of glucose. As shown in Fig. 2, a clear decrease in GSH and an increase in GSSG levels without return were observed in RBCs exposed to BHP. In this experiment, a remarkably significant inactivation of PK activity was also observed; however, GAPDH and PFK were not clearly inactivated after 1 h of incubation with BHP (Fig. 2C). After incubation of the RBC suspension for 45 min in the absence of glucose, the glucose level was less than 2 mm in RBC. Under the conditions, subsequent addition of BHP resulted in significantly decreased GSH and increased GSSG levels, which did not recover after 1 h (Figs. 3A, B). As shown in Fig. 3C, following incubation with BHP for 1 h, a significant decrease in PK activity was observed, while the activities of GAPDH and PFK were maintained despite lower levels of GSH. Effect of GSH and Glucose on Inactivation of PK Induced by BHP Exposure As shown in Fig. 4, following incubation with 0.5 mm BHP for 1 h, a marked decrease in PK activity was observed, while addition of GSH to hemolysate significantly protected PK activity in the presence of insufficient constituent including antioxidant enzymes compared to intact RBCs. However, glucose had no effect on the protection of PK activity during exposure to BHP in he- Table 2. Effects of BHP on Various Enzyme Activities in Packed RBC Enzyme Remaining activity (%) a) Control BHP b) Phosphofructokinase Glyceraldehyde-3-phosphate dehydrogenase Pyruvate kinase Hexokinase Glucose-6-phosphate dehydrogenase Glutathione peroxidase * Glutathione-S-transferase * a) Activities were expressed % (mean S.D.) for the values with incubation in the absence of BHP. b) Incubation: 2.0 mm BHP for 60 min. p 0.05 (for control). Fig. 1. Treatment of Human Red Blood Cells with BHP (A) GSH levels in RBCs exposed to 2 mm BHP (closed circles) or vehicle alone (control; open circles). (B) GSSG levels in RBCs exposed to 2 mm BHP (closed triangles). BHP was added to the RBC suspension and incubated for 60 min. A 100 ml aliquot of the RBC suspension was lysed by the immediate addition of 0.8 ml distilled water and 25% sulfosalicylic acid. After vigorous vortexing and centrifugation at 4 C and g for 5 min, the clear supernatant was used for the determination of total GSH and only GSH (reduced form) described in Materials and Methods. Concentrations of GSH and GSSG in RBCs were calculated by multiplying the value in the lysate by ten and expressed as nmol per packed cells (n 4). Representative results of three replicate experiments are shown. p 0.05.

4 1878 Vol. 31, No. 10 Fig. 2. Effects of BCNU Pre-treatment on GSH, GSSG Levels and Glycolytic Enzyme Activities Following Exposure to BHP Human RBC suspensions (Hct. 0.50) in PBS containing 10 mm glucose were incubated in the presence (closed symbol) or absence (open circles) of 1 mm BCNU for 30 min. After centrifugation to remove excess BCNU and re-suspension in PBS containing 10 mm glucose, each RBC suspension (Hct. 0.40) was treated with 2 mm BHP at 37 C for 60 min. (A) GSH concentrations (closed circles) in RBCs were determined as described in Fig. 1 (n 4). (B) GSSG concentrations (closed triangles) in RBCs were determined as described in Fig. 1 (n 4). (C) GAPDH, PFK, and PK activities within the RBCs were determined after lysing the RBC pellet as described in Materials and Methods (n 4). Representative results of duplicate experiments are shown. p 0.05, p molysate. Reactivation of PK Activity in Reconstituted Hemolysate Using the GSH-Dependent Redox System To determine whether the activity of PK is regulated by the GSH-dependent redox system in human RBCs, we utilized a system capable of inactivating and reactivating PK in hemolysate. When hemolysates were incubated with 0.25 mm BHP for 15 min, PK activity decreased by 40% was observed. When GSH was immediately added to the mixture, PK activity did not recover, as shown in Fig. 5. However, the simultaneous addition of Grx and NADPH with GSH at 15 min after BHP exposure led to a significant recovery of more than 10% PK activity. Specificity of PK Inactivation by ROS Figure 6 shows Fig. 3. Effects of Glucose Deficiency on GSH Levels and Glycolytic Enzyme Activities Following Exposure to BHP Human RBCs obtained after starvation for more than 12 h were suspended in PBS in the absence of glucose (Hct. 0.40). After incubation at 37 C for 45 min, the RBC suspensions were further incubated with BHP (closed symbol) or without (control; open circles) 2 mm BHP at 37 C. (A) GSH concentrations in RBCs were determined as described in Fig. 1 (n 4). (B) GSSG concentrations (closed triangles) in RBCs were determined as described in Fig. 1 (n 4). (C) GAPDH, PFK, and PK activities within the RBCs were determined after lysing the RBC pellet as described in Materials and Methods (n 4). Representative results of duplicate experiments are shown. p 0.05, p the residual activities in the reaction mixture after incubation with various constituents related to hydroxyl radical generating reactions. The purified PK was significantly inactivated following exposure to hydroxyl radical generated by the Fenton reaction, while a significant decrease in PK activity was not observed either with iron(iii) ascorbic acid (Asc) nor hydrogen peroxide in the reaction mixture. Over 40% of PK activity disappeared during the reaction with the hydroxyl radical generating system. Effect of GSH and Glucose on Inactivation of PK Induced by Hydroxyl Radical Exposure As shown in Fig. 7, a significant decrease of the purified PK following exposure to hydroxyl radicals was not observed in the presence of GSH. However, glucose gave no effect on the protection of

5 October Fig. 4. Effect of GSH and Glucose on PK Activity Following Exposure to BHP in Hemolysate 10% hemolysate was treated without (control) or with 0.5 mm BHP at 37 C for 60 min in the absence (BHP) or presence of 2 mm GSH (GSH BHP) or 10 mm glucose (Glc BHP). Values are expressed as means S.D. (n 4) of residual activity (unit/mg protein). Representative results of three replicate experiments are shown. Data was analyzed by analysis of variance (ANOVA). Post-hoc comparisons of means between groups were performed using Bonferroni s correction with a significance level of Not significant, p 0.01 (for control). Fig. 6. Inactivation of PK by Treatment with Various Components of the Fenton Reaction One unit/ml PK from rabbit muscle was treated with 1 mm hydrogen peroxide (H 2 O 2 ), 50 m M ascorbic acid (Asc), 50 m M Fe 3, 250 m M EDTA (Fe Asc), the complete system ( OH), or only vehicle (Blank). Following incubation for 60 min at 37 C, 20 ml of the reaction mixture was taken to evaluate the residual activity of PK. Values are expressed as means S.D. (n 4) of percent of residual activity of the control. Representative results of three replicate experiments are shown. Data was analyzed by analysis of variance (ANOVA). Post-hoc comparisons of means between groups were performed using Bonferroni s correction with a significance level of p Fig. 5. purified PK activity during exposure to hydroxyl radical generated by Fenton reaction. DISCUSSION Reactivation of PK in the Reconstituted Hemolysate System Hemolysate (10%) was treated with 0.25 mm BHP at 37 C for 15 min and aliquots were taken from the hemolysate to determine PFK activity. After taking the first sample at 15 min, 5 mm GSH (open circles) or 5 mm GSH plus 0.2 mm NADPH and 30 m M Grx (closed circles) were immediately added to the hemolysate, after which aliquots of hemolysate were taken and assayed for respective enzyme activities at the indicated time points. Values are expressed as means S.D. (n 4) of residual activity (unit/mg protein). Representative results of duplicate experiments are shown. p In the current study, we elucidated that there are differences in ROS sensitivity among thiol-dependent enzymes. We added BHP to hemolysate and RBC suspension at 0.5 mm and 2 mm based on the experiment previously reported. 6 9) HK, PFK, GST, GPX, GAPDH and PK activities could not be detected by the treatment over 1 mm BHP for 60 min in hemolysate, while no significant inactivation was observed in RBC enzymes tested by incubation in the presence of BHP under 2 mm for 60 min in RBC suspension (data not shown). Interestingly, the GSH-dependent system related enzymes are Fig. 7. Effect of GSH and Glucose on PK Inactivation by Hydroxyl Radical Produced by the Fenton Reaction One unit/ml PK from rabbit muscle was treated with 1 mm hydrogen peroxide, 50 m M ascorbic acid, 50 m M Fe 3 and 250 m M EDTA (complete system; OH) in the presence of 2mM GSH ( OH GSH) or 10 mm glucose ( OH Glc) or only vehicle (Blank). Following incubation for 60 min at 37 C, 20 ml of the reaction mixture was taken to evaluate the residual activity of PK. Values are expressed as means S.D. (n 4) of percent of residual activity of the control. Representative results of three replicate experiments are shown. Data was analyzed by analysis of variance (ANOVA). Post-hoc comparisons of means between groups were performed using Bonferroni s correction with a significance level of Not significant, p 0.01 (for control). less affected, while GST and GPX, which are capable of BHP scavenging with GSH as a substrate are more readily inactivated by BHP exposure in RBCs. Inactivation of GPX by superoxide 23) or by interaction with oxidized hemoglobin in human RBCs have been reported. 24,25) Our results indicate that GST and GPX activity may be utilized as oxidative stress markers in vivo. It is likely that the best examples are glycolytic pathway proteins that are functionally activated or inactivated during oxidative stress. 10,11) In general, GSH and antioxidant enzymes capable of scavenging oxidants, such as hydrogen peroxides, contribute to regulating the redox status

6 1880 Vol. 31, No. 10 of the cell. The GSH-dependent redox system, which utilizes Grx, G6PDH, and GR activities, is more functional in humans than in other animal species. 4,26) Indeed, large amounts of GSH-mixed disulfides are produced in rats, but not in human RBCs, following treatment with hydroperoxides or diamide. 8) However, it is still unclear which protected thiol residues depend on GSH in human RBCs. To elucidate the real contribution of GSH in the protection of protein thiol residues in human RBCs, we investigated changes in various enzyme activities following BHP exposure in GSH homeostasis. It was clear from this study that glycolitic and related enzymes were susceptible to oxidation. Further, GSH seems to protect all thiol-dependent enzymes because oxidative inactivation of thiol-dependent enzymes, including HK, G6PDH, GAPDH, PFK, and PK, was not observed following exposure of an intact RBC suspension to BHP. Thus, we first examined the changes in thiol-dependent enzymes following BHP exposure and disruption of the GSH homeostasis, as well as alterations in GSH and GSSG to show the contribution of GSH to the regulation of protein thiol residues in RBCs. It was clearly shown that reductive GSH levels (GSH/GSSG ratio) are strictly regulated in intact RBCs. This result strongly indicates the essentiality of GSH homeostasis and its maintenance mechanism orderly act in RBC suspensions with glucose. As shown in the results presented here, two independent pre-treatments aimed at disrupting GSH homeostasis caused accumulation of GSSG in responses of RBCs to BHP. Also, it is possible that the modification of protein-sh groups is occurred via the reaction with GSSG in RBCs. It appeared that GSH disruptive conditions produced by pre-treatment with an inhibitor of GR or incubation in glucose-free medium are dependent on the regulatory balance between glycolysis and the pentose phosphate pathway. In the case of glucose deficiency, the pentose phosphate pathway is limited in reproducing NADPH that is required for the GSH-dependent redox system. Under these conditions, we examined the direct or indirect antioxidant roles of GSH in preventing the protein-sh groups. We measured alterations in PFK, GAPDH, and PK activities because the glycolytic pathway proteins are functionally activated or inactivated during oxidative stress. 10,11) Interestingly, a clear decrease in PK activity was observed during the disruption of GSH homeostasis, while remarkable changes in GAPDH and PFK activities were not observed. GSH seems to react directly with hydroxyl radicals 27,28) and it was reported that BHP produces t-butyl hydroxyl radicals in human RBCs. 7) On the other hand, PK is an enzyme that is readily inactivated by ROS. 29) However, it remains unclear whether the GSH-dependent system contributes to scavenge radicals and protect enzymes in vivo. Further, the sensitivity of PK to radical oxygen species is obscure. Thus, our present study shows the first evidence that GSH protects PK activity in intact RBCs and that PK is highly susceptible to BHP, which is capable of generating radicals in RBCs. In addition, PK inactivated by BHP exposure could be reactivated by the GSH-dependent system at an early stage of oxidation, while non-enzymatic reactivation by GSH did not occur. These results suggest that PK is initially oxidized to a reversible form which is reactivated by GSH-dependent redox system. In this study, clear inactivation of PFK and GAPDH was not observed with BHP exposure in GSH-depleted RBCs. These results indicate that the protection of PFK and GAPDH activities may be regulated at low levels of GSH or that their reactivities to ROS are lower than PK in intact RBCs. On the other hands, it is possible that GSH depletion limits the role of GPX and GST to scavenge BHP resulting in acceleration of inactivation on PK activity in RBCs. This possibility of substrate depletion for GPX and GST is also involved in the effect caused by disruption of GSH homeostasis. Anyway, it is likely that there are differences in the susceptibility of PK and the other two enzymes against ROS exposure. To elucidate the mechanism of inactivation of PK, we examined the sensitivity of PK to radicals using purified PK from rabbit muscle. Our results show that PK has highly susceptible site to radical species but not peroxides. Moreover, it was clearly shown that GSH directly protect the PK activity against hydroxyl radical (see Fig. 7). Although it has been indicated that inactivation of rabbit muscle PK was occurred primarily due to modification of cysteine residues in the active site, 30,31) there are little evidences of human PK. 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