erythrocyte membranes (transport/inhibition/isozyme)

Transcription

1 Proc. Nad. Acad. Sci. USA Vol. 84, pp , November 1987 Biochemistry Glutathione disulfide-stimulated Mg2+-ATPase of human erythrocyte membranes (transport/inhibition/isozyme) TAKAHITO KONDO*, YOSHIKAZU KAWAKAMI*, NAOYUKI TANIGUCHIt, AND ERNEST BEUTLER* *First Department of Medicine, Hokkaido University School of Medicine, Sapporo 060, Japan; tdepartment of Biochemistry, Osaka University School of Medicine, Osaka 530, Japan; and *Department of Basic and Clinical Research, Research Institute of Scripps Clinic, La Jolla, CA Contributed by Ernest Beutler, June 5, 1987 ABSTRACT Inside-out erythrocyte membranes attached to polycationic beads manifested glutathione disulfide (GSSG)- stimulated ATPase activity. A Lineweaver-Burk plot of the ATPase activity as a function of GSSG concentration was biphasic and gave apparent K. values of 0.13 mm and 2.0 mm. These kinetics are similar to those reported for the ATPrequiring GSSG-transport systems in human erythrocytes and for the GSSG-stimulated ATPase activity in the plasma membranes of rat hepatocytes. Erythrocyte membranes that were depleted of extrinsic proteins were solubilized in 0.5% Triton X-100. Affinity chromatography on S-hexylglutathione-Sepharose 6B, with elution by a linear gradient of S-hexylglutathione, resulted in the resolution of two peaks of enzyme activity. One enzyme, which was eluted at -0.5 mm S- hexylglutathione, had a high affminty for GSSG (apparent K. of 150,uM) and for ATP (80,uM). The other enzyme, which was eluted at =1 mm S-hexylglutathione, had a low affinity for GSSG (apparent K. of 2.0 mm) and ATP (140 pm). GSSGindependent Mg2+-ATPase, Ca2+-dependent Mg2+-ATPase and Na+,K+-dependent Mg2+-ATPase were undetectable in the fractions. Addition of Ca2, ouabain, or vanadate neither activated nor inhibited the activities, further indicating that the enzymes are distinguishable from ion-pumping ATPases. The enzymes required GSSG for activation; reduced glutathione (GSH) was ineffective. The ATPase activity of the high-km enzyme was inhibited by addition ofp-chloromercuribenzoate, N-ethylmaleimide, and iodoacetamide and was activated by treatment with dithiothreitol, whereas the ATPase activity of the low-km enzyme was not modified by these thiol reagents. The properties of the enzymes are similar to those of ATPdependent GSSG-transport systems in human erythrocytes, suggesting that these ATPases may function in the active transport of GSSG. Glutathione is synthesized in erythrocytes with a turnover rate of -7 nmol of oxidized glutathione (GSSG) per hr per ml of erythrocytes (1). Active, ATP-requiring transport ofgssg from human erythrocytes was initially demonstrated after increasing the intracellular GSSG concentrations by oxidative stress (2). The GSSG-transport system was subsequently investigated using resealed membranes (3, 4). The rate of this transport was shown to be dependent on the intracellular GSSG levels (5). The rate of GSSG transport from erythrocytes under physiological conditions was found to correspond to the amount of glutathione synthesized (6). Furthermore, we reported that conjugates of glutathione with electrophilic compounds are transported competitively with the GSSG (7). However, the precise mechanisms by which ATP is involved in the GSSG transport have not been clarified, and the existence of a GSSG-requiring ATPase could not be demonstrated. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. Recently, Nicotera et al. (8) showed that GSSG-stimulated ATPase activity is present in plasma membranes from rat hepatocytes. In the present study, we investigated the GSSGdependent enzymatic hydrolysis of ATP in human erythrocyte membranes. MATERIALS AND METHODS Preparation of Erythrocytes. Fresh human venous blood was collected from normal donors into a 1-mg/ml solution of ethylenediaminetetraacetic acid (EDTA) and filtered through a small column of a-cellulose-microcrystalline cellulose to remove leukocytes and most platelets (9). The erythrocytes were washed twice in M NaCI. Attachment of Erythrocyte Membranes to Polycationic Beads. Erythrocyte membranes were ionically attached to polyethyleneimine-coated beads (Affi-Gel 731, Bio-Rad) as described by Jacobson and Branton (10). In brief, 2 g of the beads were washed three times with 0.2 M NaCl, twice with 150 mm Tris*HC1 buffer (ph 7.4), and once with buffer A (7 volumes of 310 mm sucrose and 3 volumes of 49 mm sodium acetate, ph 5.0). Eight milliliters of a 50o (vol/vol) suspension of beads was added slowly to 4 ml of washed erythrocytes suspended in buffer A. After washing with buffer A twice, hemoglobin was removed from erythrocytes attached to the beads by washing three times in 30 volumes of 10 mm Tris-HCl (ph 7.4) at 0C, and the cells were disrupted by sonication for 5 sec. The membrane/bead suspension, washed with Tris*HCI (ph 7.4), was resuspended in the same buffer and kept at 0 C until use. Purification of GSSG-Stimulated ATPases. All subsequent procedures were performed at 4 C. Essentially hemoglobinfree erythrocyte membranes ("white ghosts") were prepared by lysing 100 ml of the washed erythrocytes in 30 volumes of 10 mm Tris HCI (ph 7.4), and washing five times in the same buffer (4). White ghosts were suspended in 20 volumes of 0.5 mm Tris-HCl (ph 8.0) to remove most of the extrinsic proteins such as spectrin (11). Spectrin-depleted ghosts (80 ml) were collected by centrifugation at 15,000 x g for 30 min and were solubilized in 0.5% (wt/vol) Triton X-100/10 mm imidazole HCI, ph 7.4/0.2 mm EDTA/50,uM 2-mercaptoethanol (buffer B), for 20 min. Insoluble proteins were removed by centrifugation at 100,000 X g for 90 min. Seventy-five milliliters of the extract was diluted with an equal volume of 10 mm imidazolehci, ph 7.4/0.2 mm EDTA/50,AM 2-mercaptoethanol (buffer C) and applied to a column of S-hexylglutathione-Sepharose 6B (1 x 6 cm) that had been equilibrated with 0.25% Triton X-100 in buffer C. The column was washed with 50 ml of 0.5 M NaCI in buffer C, and bound material was eluted with a 60-ml linear gradient Abbreviations: GSH, reduced glutathione; GSSG, oxidized glutathione (glutathione disulfide); 'Cys'-SSG, glutathione cysteine mixed disulfide; Glu('Cys'-SSG), glutathione y-glutamylcysteine mixed disulfide. 7373

2 7374 Biochemistry: Kondo et al. Proc. Natl. Acad. Sci. USA 84 (1987) of 0-2 mm S-hexylglutathione at a flow rate of 8 ml/hr. Fractions (1 ml) of the eluent were collected, and enzyme activities and protein concentration of fractions were determined. GSSG-Stimulated Mg2+-ATPase Activity. Unless otherwise indicated, GSSG-stimulated ATPase activity was measured at 370C in a 250-,ul system containing 100 mm Tris HCl (ph 7.4), 0.5 mm EDTA, 10 mm MgCI2, 1 mm ATP, 5 mm or 50,uM GSSG, and 150 Al of enzyme solution (2-600,g of protein). The reaction was terminated after 30 min by the addition of 250 A1 of ice-cold 8% tricholoroacetic acid. When the protein content in the assay system was <20,ug, 100,g of bovine serum albumin was added after termination of the reaction. The tubes were centrifuged in the cold, and inorganic phosphate (Pi) was extracted from the supernatant into benzene/isobutanol (1:1, vol/vol) as a molybdate complex (12) and was assayed colorimetrically as described by Martin and Doty (13). The activity was taken as Pi released by the addition of GSSG. As a blank system, no GSSG was added to the assay system. In some studies of GSSG-stimulated ATPase, [y-32p]atp with a specific activity of 1.5 pmol/dpm and [8-3H]ATP with a specific activity of 1.6 pmol/dpm were used. Ca2+-dependent or Na+,K+-dependent Mg2+-ATPase activities were assayed as described (14). ATP labeled with 32p in the y position was synthesized enzymatically as described (15). [3H]ATP labeled in the 8 position was obtained from New England Nuclear. Radioactivity of 32P and 3H was measured in a liquid scintillation counter. Chromatographic separation of Pi from nucleotides was achieved by applying the sample to a Dowex-1 column (2.2 x 25 cm) and eluting with a 500-ml linear gradient of M HCO according to the established method (16). Elution positions of AMP, ADP, and ATP in this chromatographic system were established using pure samples (Sigma). Protein Determination. Protein concentrations were determined according to the method of Lowry et al. (17), with bovine serum albumin as a standard. Electrophoresis. NaDodSO4/PAGE was performed using Phast Gel (gradient, 8-25% acrylamide) and a Phast System (Pharmacia). Molecular weight standards for NaDodSO4/ PAGE were purchased from Bio-Rad. Preparation of Glutathione Analogues. Glutathione y- glutamylcysteine mixed disulfide [Glu('Cys'-SSG)] and glutathione cysteine mixed disulfide ('Cys'-SSG) were synthesized as described (18, 19). S-Hexylglutathione was synthesized according to the method of Mannervik and Guthenberg (20). S-Hexylglutathione-Sepharose 6B was prepared as described (20), using epoxy-activated Sepharose 6B (Pharmacia). 7 E a) 0.8 C.E " 0.6- a) CL Ef 0.4- > 0.2- = 2.0 mm (GSSG concentration)-1, (mm)-' FIG. 1. Lineweaver-Burk plot of GSSG-stimulated ATPase activity (nmol of Pi released per min per mg of protein) of erythrocyte membranes attached to polycationic beads, as a function of GSSG concentration. Membrane/bead suspension (100,l) was incubated with 1 mm ATP, 10 mm MgCl2, 250,ul of 100 mm Tris HCl (ph 7.4) and various concentrations of GSSG for 30 min at 37 C. Incubation system without GSSG served as blanks. RESULTS Identification of GSSG-Stimulated ATPase in Erythrocyte Membranes. In preliminary studies using erythrocyte ghosts, activities of ATPases tended to decrease in the presence of GSSG, suggesting that GSSG-stimulated ATPase activities were masked by the inhibitory effect of GSSG on other ATPases. In the absence of Mg2", addition of GSSG to the membranes attached to polycationic beads resulted in hydrolysis of ATP. In the presence of 1 mm GSSG and 1 mm ATP, the amount of Pi hydrolyzed from ATP was approximately twice that observed in the absence ofgssg. Pi release was dependent on incubation time at 37 C for up to 3 hr. Addition of GSSG to the beads without attached erythrocyte membranes did not result in ATP hydrolysis. The dependence of GSSG concentration on GSSG-stimulated ATPase activity was measured over a 25,.M-10 mm range of GSSG. Lineweaver-Burk analysis of these data gave a biphasic plot with two apparent Km values of 0.13 mm and 2.0 mm GSSG, having apparent Vmax values of 6.4 and 38.5 nmol of Pi released per min per mg of protein, respectively (Fig. 1). Partial Purification of GSSG-Stimulated ATPases. In order to estimate activities of the putative distinct GSSG-stimulat- Table 1. Preparation of low-km and high-km forms of GSSG-stimulated ATPase from human erythrocytes GSSG-stimulated Specific activity, Volume, Protein, ATPase, nmol of Pi nmol of Pi released Purification Yield, Step ml mg released per min per min per mg of protein factor % Low-Km form Spectrin-depleted ghosts Solubilization in 0.5% Triton X Affinity chromatography on S-hexylglutathione- Sepharose 6B High-Km form Spectrin-depleted ghosts Solubilization in 0.5% Triton X Affinity chromatography on S-hexylglutathione- Sepharose 6B Kml = 0.13 mm

3 Biochemistry: Kondo et al. Proc. Natl. Acad. Sci. USA 84 (1987) 7375 a_ C. U) CU I- T Ẹ ) 0 0 a. CD C') LI) CO, FIG. 2. Fraction Affinity chromatography of two GSSG-stimulated ATPases. Erythrocyte membranes solubilized in 0.25% Triton X-100/10 mm imidazole HCI, ph 7.4/0.2 mm EDTA/50 AtM 2-mercaptoethanol were chromatographed on S-hexylglutathione-Sepharose 6B. Elution was with a 0-2 mm S-hexylglutathione gradient. The low-km form of GSSG-stimulated ATPase activity is represented by open circles and the high-km form by the filled circles; activity is expressed as nmol of Pi released per min per ml. Protein concentration is represented by the open bars. ed ATPases, 50,uM and 5 mm GSSG concentrations were used to detect the low-km and high-km enzymes, respectively. Until the step of solubilization of membrane proteins by Triton X-100, GSSG-stimulated ATPase activities were estimated as a net increase of Pi released by 50 ILM GSSG or 5 mm GSSG at an initial ATP concentration of 1 mm in the absence of added Mg2". GSSG-stimulated ATPase activities in the fractions obtained by affinity chromatography on S-hexylglutathione-Sepharose 6B were estimated in the presence of 10 mm Mg2". As shown in Table 1, a 2500-fold purification of the low-km enzyme was achieved relative to the activity of spectrin-depleted erythrocyte membranes. The overall yield of the enzyme activity was 71%. Fig. 2 shows the elution profile from S-hexylglutathione-Sepharose 6B. The low-km enzyme was eluted at =0.5 mm S-hexylglutathione and the high-km enzyme at -1 mm S-hexylglutathione. The overall yield of enzyme activity was 35.1%. Purification of the high-km enzyme was 1054-fold and the overall yield of enzyme activity was 33% (Table 1). No GSSG-independent ATPase activity was detectable in active fractions in the final step of purification of the two enzymes. Active fractions in buffer C were rapidly frozen in liquid nitrogen and stored at -80TC until use for the following experiments. Electrophoresis. The electrophoretic profile of the two enzymes showed similar silver-staining protein bands. The two major bands corresponded to molecular weights of approximately 65,000 and 85,000, as determined by comparison of their mobilities with those of the standards run in the same slab gel. Characterization. Some properties of the enzymes were investigated. Since the partially purified fractions contained no GSSG-independent ATPase, assay mixtures with 1 mm ATP, 10 mm MgCI2, and either 50,uM GSSG (low-km enzyme) or 5 mm GSSG (high-km enzyme) were used; the blank system was composed of the same components but without the enzyme fractions. Effect of incubation time on Pi release was linear for 3 hr at 1 mm ATP with both enzymes. The activities of the enzymes were dependent on concentrations of GSSG and Mg-ATP. The low-km enzyme had apparent Km values of 150,uM for GSSG and 80,uM for Mg-ATP, and the high-km enzyme, 2.0mM for GSSG and 140 1LM for Mg-ATP. Neither enzyme fraction manifested Ca2+dependent or Na',K+-dependent Mg2+-ATPase activity. Effect of Various Inhibitors and Chelators. The effect of EDTA, EGTA, ouabain, and vanadate on the enzyme activities was studied. The enzymes required Mg2' and lost activities when 5 mm EDTA was added. Addition of 5 mm EGTA, 1 mm Ca2+, 30,uM vanadate, or 0.1 mm ouabain did not show any effect on the activities. These results indicate that the enzyme activities are independent of Ca2+, Na+, or K+ and are quite distinct from the ATPases that serve to transport these ions. Substrate Specificities. The enzymes did not hydrolyze ADP, AMP, or adenosine 5'-[,8,y-imido]triphosphate. Specificity of substrates other than GSSG was studied (Table 2). The assay systems employed were the same as for GSSG. The mixed disulfides Glu('Cys'-SSG) and 'Cys'-SSG partially replaced the requirement of the ATPase activities for GSSG, whereas reduced glutathione (GSH) had no effect. Effect of Thiol Reagents. Preincubation of high-km enzyme with 1 mm dithiothreitol, followed by passage through a PD-10 column to remove excess dithiothreitol, resulted in an activation of the enzyme activity. Addition of thiol reagents such as p-mercuribenzoate, N-ethylmaleimide, or iodoacetamide decreased the activity of the high-km enzyme (Table 3). Thiol reagents had no apparent effect on the activity of the low-km enzyme. Identification of the Reaction Products. The products of ATP cleavage were investigated by using a mixture of [3H]ATP and [y-32p]atp as substrate. After incubation of purified enzyme solution with 0.1 mm doubly labeled ATP for 30 min at 370C, the reaction was terminated by the addition of 0.1 volume of 80% trichloroacetic acid, and 0.07 volume of 5% bovine serum albumin was then added. The diethyl ether-extracted supernatant was chromatographed on Dowex-1 (Cl-) (Fig. 3). 32P activity was found in the Pi peak, eluted at M HCI, in the ATP peak, eluted at 0.1 M HCI, and in the ADP peak, eluted at 0.05 M HCL. The amount of Pi and ADP formed corresponded to the decreased amount of ATP. No other peak of 32p or 3H activity was detected. There was no difference in the chromatographic profiles produced Table 2. Substrate specificity of GSSG-stimulated ATPases Concentration, Activity,* % Substrate mm LOw-Km High-Km GSSG GSH 'Cys'-SSG Glu('Cys'-SSG) *Expressed as a mean of two experiments with duplicate analysis.

4 7376 Biochemistry: Kondo et al. Table 3. Effect of thiol reagents on GSSG-stimulated ATPase activity Concentration, Activity,* % Reagent mm Low-Km High-Km None Dithiothreitol p-chloromercuribenzoate N-Ethylmaleimide lodoacetamide Enzyme was preincubated with the indicated reagents and was assayed after gel filtration to remove excess reagent. *Expressed as a mean of two experiments with duplicate analysis. by the two enzymes, indicating that both enzymes catalyze the hydrolysis of ATP to form ADP and Pi. DISCUSSION Outward transport of GSSG from erythrocytes requires ATP. We previously reported the characterization of two independent GSSG transport systems (3, 4). The transport velocity is dependent on Mg-ATP concentration. Therefore it seemed likely that the transport systems would include GSSGstimulated ATPases. Since erythrocytes are very rich in various types of ATPase activity, we were initially unable to identify a GSSG-dependent enzymatic activity. it became possible to observe GSSG-stimulated ATPase activity by using inside-out plasma membranes attached to polycationic beads without the addition of Mg2+ (8). In this way we were.1) 0 x E 'D N 0: C0 co X Q I AMP Pi I I sr"eqm. QPGW ADP I ATP I, R%9.0999owl- 0o w-- 0 ROA ATP I ---:- '. ---,m I I I 0 10 io2 30o 40 do io 7 o Fraction FIG. 3. Dowex-1 (Cl-) chromatography of the end products ofthe high-km form of GSSG-stimulated ATPase after incubation with [3H]ATP and [y-32p]atp. The reaction system contained 0.1 mm ATP, 0.1 M Tris-HCl (ph 7.4), 1 mm GSSG, 0.5 mm EDTA, 2 mm MgCl2, and either GSSG-stimulated ATPase (o) or a blank without the enzyme (.). The reaction was stopped with 0.1 volume of 80% trichloroacetic acid followed by 0.07 volume of 5% bovine serum albumin, the mixture was extracted with diethyl ether, and the extract was chromatographed on a 2.2 x 25-cm Dowex-1 (Cl-) column with a M HCI gradient. Fractions (12 ml) were collected, and 32p (Upper) and 3H (Lower) were measured by liquid scintillation counting. I Proc. Natl. Acad. Sci. USA 84 (1987) able to identify the enzyme activity in erythrocyte membranes. A biphasic Lineweaver-Burk plot of the activity of membranes/beads was observed as a function of GSSG concentration, suggesting the presence of two different GSSG-stimulated ATPases. Since the partially purified enzymes required Mg2+ for activity, it is suggested that GSSG-stimulated ATPases of the plasma membranes utilize Mg2+ intrinsic to the membrane. The two enzymes were partially prepared by solubilization of erythrocyte membranes with Triton X-100 followed by affinity chromatography on S-hexylglutathione-Sepharose 6B. The low-km enzyme was eluted at 0.5 mm S-hexylglutathione, and the high-km enzyme, at 1 mm fold purification of the low-km enzyme and an 1000-fold purification of the high-km enzyme were achieved, and further characterization was done using these partially purified enzymes. They had no ATPase activity in the absence of GSSG, but they cleaved ATP in the presence of this disulfide. The enzyme activities were inhibited by the addition of EDTA but not by the addition of ouabain, and the addition of Ca2, vanadate, or EGTA had no effect on their activities. They showed no Na+,K+-dependent Mg2+-ATPase activity or Ca2'-dependent Mg2+-ATPase activity. It is clear that GSSG-stimulated Mg2+-ATPases are distinct from these ion-pumping ATPases. Since the GSSG-dependent enzymes require Mg2' and hydrolyze ATP to form ADP and Pi, they also clearly differ from nonspecific neutral phosphatases. GSSG transport is achieved by erythrocytes by two independent systems, as indicated by different kinetic parameters, ph optima, temperature dependence, effect of storage, or effect of thiols (4). The high-ki, but not the low-km, transport system is activated by treatment with dithiothreitol and inhibited by the addition of p-chloromercuribenzoate, iodoacetamide, or N-ethylmaleimide. The high-km enzyme prepared in this study was activated by the addition of dithiothreitol and inhibited by the addition of thiol reagents, whereas these compounds had no effect on the low-km enzyme. Furthermore, substantial stimulation of the enzymes was observed with disulfides ['Cys'-SSG and Glu('Cys'-SSG)] other than GSSG, but no stimulatory effect was observed with GSH. These properties resemble those of the GSSG-transport systems. It therefore seems reasonable to conclude that these enzymes play a role in the outward transport of GSSG in human erythrocytes. This work was supported in part by National Institutes of Health Grant HL This is paper no. 4890BCR from the Research Institute of Scripps Clinic. 1. Dimant, E., Landberg, E. & London, I. M. (1955) J. Biol. Chem. 213, Srivastava, S. K. & Beutler, E. (1969) J. Biol. Chem. 244, Kondo, T., Dale, G. L. & Beutler, E. (1980) Proc. Natl. Acad. Sci. USA 77, Kondo, T., Dale, G. L. & Beutler, E. (1982) Biochim. Biophys. Acta 645, Prchal, J., Srivastava, S. K. & Beutler, E. (1975) Blood 46, Lunn, G., Dale, G. L. & Beutler, E. (1979) Blood 54, Kondo, T., Murao, M. & Taniguchi, N. (1982) Eur. J. Biochem. 125, Nicotera, P., Moore, M., Bellomo, G., Mirabelli, F. & Orrenius, S. (1985) J. Biol. Chem. 260, Beutler, E., West, C. & Blume, K. G. (1976) J. Lab. Clin. Med. 88, Jacobson, B. S. & Branton, D. (1976) Science 195, Steck, T. & Kant, L. 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