Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes
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1 Eur. J. Biochem. 145, (1984) 0 FEBS 1984 Significance of glutathione S-conjugate for glutathione metabolism in human erythrocytes Takahito KONDO, Naoyuki TANIGUCHI, and Yoshikazu KAWAKAMI First Department of Medicine, and Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Sapporo (Received April 24/August 2, 1984) - EJB The significance of glutathione S-conjugate in the regulation of glutathione synthesis was studied using human erythrocyte y-glutamylcysteine synthetase. Feedback inhibition of the enzyme by reduced glutathione was released by the addition of the glutathione S-conjugate (S-2,4-dinitrophenyl glutathione). A half-maximal effect of glutathione S-conjugate on y-glutamylcysteine synthetase activity was obtained at approximately 1 pm; 50 pm glutathione S-conjugate in the presence of 10 mm glutathione actually increased the enzyme activity twofold above uninhibited levels. Glutathione S-conjugate had no effect on the enzyme activity in the absence of glutathione. When erythrocytes were exposed to the electrophile l-chloro-2,4-dinitrobenzene, which forms a glutathione S-conjugate by the catalytic reaction of glutathione S-transferase, the level of glutathione synthesis increased. These data suggest that glutathione S-conjugate plays a role in stimulating the synthesis of glutathione. Glutathione exists in virtually all living cells in a reduced form (GSH) and an oxidized form (GSSG). It has been demonstrated that the biosynthesis of GSH is catalyzed by two enzymes [l]: y-glutamylcysteine synthetase (GC synthetase) and glutathione synthetase. ~-Glutamic acid + L-cysteine + ATP $:+ y-gluta- myl-l-cysteine + ADP + Pi. y-glutamyl-l-cysteine + glycine + ATP g ' u ~ ~ ~ ~ ~ ~ t a s e GSH + ADP + Pi. In human erythrocytes GSH is synthesized by these two enzymes [2]. GSH forms S-conjugates with a number of electrophilic compounds, including various drugs and xenobiotics, through a reaction catalyzed by glutathione S-transferase (GSH S- transferase) [3]. GSH S-transferase is also thought to be important as a binding protein for the solubilization [3] and transport of electrophilic or xenobiotic chemicals [4]. An isozyme designated as GSH S-transferase p has been purified from human erythrocytes. Its immunological characteristics are different from those of the multiple forms of GSH S- transferase in human liver [5]. The erythrocyte enzyme is thought to be important for the removal of circulating xenobiotics [5]. Furthermore Harvey and Beutler have recently reported that GSH S-transferase functions physiologically as a hemin-binding and/or transport protein in developing erythroid cells [6]. The formation of GSH S-conjugates is accompanied by a decrease in GSH levels. Erythrocyte GSH can be depleted Abbreviations. GSH, reduced glutathione; GSSG, oxidized glutathione; GSH S-transferase, glutathione S-transferase; GC synthetase, y-glutamylcysteine synthetase; GS-N2ph, S-2,4-dinitrophenyl glutathione; C1N2Bz, 1 -chloro-2,4-dinitrobenzene. Enzymes. Glutathione S-transferase (EC IS); y-glutamylcysteine synthetase (EC ). (1) completely by incubating the cell with 1 -chloro-2,4- dinitrobenzene (C1N2Bz), which is a substrate for GSH S- transferasep [7]. The ClN2Bz reacts with GSH to form S-2,4- dinitrophenyl glutathione (GS-N2ph). Quite recently Bilzer et al. reported that GSH S-conjugate interacts with glutathione reductase in hepatocytes from rats IS]. This indicates that GSH S-conjugate may alter the mechanisms by which GSH synthesis is regulated. In the present study we have demonstrated that GSH S-conjugate increases the activity of GC synthetase inhibited by GSH. MATERIALS AND METHODS Chemicals a-cellulose, microcrystalline cellulose, sodium ATP, GSH, and L-a-aminobutyric acid were purchased from Sigma Chemical Co. DEAE-cellulose (DE-52) and Sephadex G-150 were from Pharmacia Fine Chemicals. ClN2Bz was from J. T. Baker Chemical Co. Dowex-I was from Bio-Rad Laboratories. Other chemicals used were of analytical grade. Isolation of GC synthetase GC synthetase was partially purified from human erythrocytes according to the method described [2]. Briefly, fresh blood from normal donors was collected in a final concentration of 0.27 mm EDTA and was freed of leucocytes and platelets as described [9]. Erythrocytes were hemolyzed by the addition of 10 vol. cold distilled water followed by centrifugation to remove stroma. Hemoglobin in the hemolysate was removed batchwise using DEAE-cellulose equilibrated with 3 mm sodium phosphate buffer, ph 7.0. The eluate was fractionated by ammonium sulfate (45-75%) and then applied to a column of DEAE-cellulose equilibrated with 20 mm sodium phosphate buffer, ph 7.0, followed by fractionation on Sephadex G-150 and aminohexyl-atp-sepharose 4B [lo].
2 132 Pur ijica tion of GSH S - trans ferase An anionic form of GSH S-transferase was purified from human liver as described [l 11 using an affinity chromatography column prepared by coupling GSH to epoxy-activated Sepharose 4B [12]. Preparation of GSH S-conjugate GS-Nzph was prepared enzymatically as described [13], using 3 mm GSH, 4 mm CINzBz, and 1 unit of GSH S- transferase. GS-Nzph prepared using Dowex-1 (formate form) was evaporated using a rotary evaporator. Traces of formic acid were removed as follows. Sediments of GS-Nzph were suspended in 0.5 ml of cold acetone, and 2 ml of hexane was added to the suspension. After centrifugation at 2000 x g for lomin, the supernatant was dried under Nz gas. The conjugate was stored at - 20 C until use. The concentration of GS-Nzph was estimated photometrically at 340 nm as described by Wahllander and Sies [14]. Other conjugates, p- nitrobenzyl glutathione and p-nitrophenyl glutathione, were prepared using p-nitrobenzyl chloride and p-nitrophenyl acetate, respectively, according to the method employed for the preparation of GS-Nzph. [3H]GSH was used to estimate the concentration of the conjugates. Methods Enzyme activity of GC synthetase was determined essentially as described [lo]. Unless otherwise indicated, the reaction mixture consisted of 0.1 M Tris/HCl, ph 8.2, 10 mm L-glutamic acid, 10 mm L-a-aminobutyric acid, 2 mm EDTA, bovine serum albumin (40 pg/ml), 20 mm MgClZ, 5 mm ATP, and enzyme (3 units) in a final vol. of 0.5 ml. In order to observe the effect of GSH or GS-Nzph on the enzyme activity, the reaction mixture was preincubated with them for 15 min at 37 C and the reaction was initiated by the addition of L-Maminobutyrate. The assay mixture was incubated at 37 C for 15 min, and the reaction was terminated by the addition of an equal volume of 10% trichloroacetic acid. The Pi released was estimated at 720 nm using a Shimadzu electrophotometer MPS 2000 by the method of Martin and Doty [15]. One unit of the enzyme activity was defined as the catalytic activity corresponding to 1 pmol Pi released/h. The formation of L-yglutamyl-L-a-aminobutyrate by the catalytic reaction of GC synthetase was also determined using ~-a-['~c]aminobutyrate as a substrate according to the method described [16]. ATPase activity in the absence of amino acids was determined by the formation of Pi as described [17J Biosynthesis of GSH in erythrocytes Biosynthesis of GSH in red cells was determined as described [18]. Briefly, 1 ml of fresh erythrocytes free of leucocytes and platelets was incubated at 20% hematocrit for 4 h at 37 C in the presence or absence of 0.4 mm CINzBz, in 1 % bovine serum albumin, 8 mm glucose, 62 mm NaC1, 40 mm NaHzPO4/Na2HPO4, 35 mm Na/Tes ph 7.4 with 5 yci [3H]glycine (23 yci/nmol). After incubation the erythrocytes were washed to remove most of the residual [3H]glycine. The cell suspension was lysed by three freezethaw cycles and treated with t-butylhydroperoxide to oxidize GSH to GSSG. The hemolysate was centrifugated, and the supernatant was dialyzed against 100 ml HzO. The outer solution, with a carrier of GSSG and GS-Nzph, was applied to a Dowex-I column (formate form). The column was eluted with a 50-ml linear gradient of 0-4 M formate. The concentrations of carrier GSSC and GS-Nzph in each fraction were assayed as described [14, 191. The concentration of [3H]GSSG and [3H]GS-Nzph was determined using a liquid scintillation counter. RESULTS Inhibition of GC synthetase by GSH and compounds related to GSH Table 1 shows the effect of GSH and GSH S-conjugates on GC synthetase activity. The enzyme isolated from human Table 1. Effect of glutathione and its related compounds on GC synthetase from human erythrocytes The reaction mixture was composed of 0.1 M Tris/HCl, ph 8.2, 10 mm L-glutamate, 10 mm L-a-aminobutyrate, 2 mm EDTA, 0.4% bovine serum albumin, 20 mm MgCI2, 5 mm ATP and 3 units enzyme in a final volume of 0.5 ml. The assay mixture was incubated at 37 C for 15 min. Values are expressed as an average f SD of two experiments with duplicate assays for each Reaction mixture Pi released Activity L-Glutamyl remaining aminobutyrate formed pmol Yo pmol Control 0.72 & 0.01 (100) 5 mm GSH 0.33 k 0.03 (46) 0.68 f f mm GSH 0.21 * 0.02 (29) 0.20 f pm GS-Nzph 0.72 f 0.01 (100) 0.67 f mm C1N2Bz 0.72 f 0.01 (100) 0.67 F pm p-nitrobenzyl glutathione (100) pm p-nitrophenyl glutathione 0.72 k 0.01 (100) 0.67 f mm GSH + 1 pm GS-Nzph 0.47 & 0.01 (65) 0.45 f mm GSH + 1 pm GS-N2ph 0.30 f 0.02 (42) 0.28 f mm GSH + 1 pm p-nitrobenzyl glutathione 0.31 & 0.02 (43) 0.27 * mm GSH + 1 pm p-nitrophenyl glutathione 0.30 f 0.01 (42) mm L-y-glutamyl-L-cysteine 0.52 f 0.01 (72) 5 mm L-y-glutamyl-L-cysteine 0.36 & 0.02 (50) 0.48 f f mm L-y-glutamyl-L-cysteine + 1 pm GS-N2ph 0.52 * 0.02 (72) 0.49 f mm L-y-glutamyl-L-cysteine + 1 pm GS-Nzph 0.36 * 0.01 (50) 0.34 f mm GSSG 0.68 f 0.02 (94)
3 erythrocytes was inhibited to 46% and 29% of the original activity in the presence of 5 mm and 10 mm GSH, respectively. However GSSG had no significant effect on the enzyme activity. y-glutamyl-l-cysteine, which is the product synthesized by GC synthetase, also inhibited the enzyme. Release of the inhibitory effect of GSH on GC synthetase with the addition of GSH S-conjugates As shown in Table 1, addition of 1 pm GS-N2ph resulted in an increase in enzyme activity in the presence of 5 mm and 10mM GSH from 46% to 65% and from 29% to 42%, respectively. GS-N2ph or ClN2Bz had no direct effect on the enzyme activity in the absence of GSH. Addition of other GSH S-conjugates, p-nitrobenzyl glutathione or p-nitrophenyl glutathione, to the reaction mixture resulted in the increase of the GC synthetase activity similar to GS-N,ph. These GSH S-conjugates also had no direct effect on the enzyme activity in the absence of GSH. In order to discover whether there were other phosphate-hydrolyzing activities in the enzyme whch are not associated with the formation of L- y-glutamyl-l-a-aminobutyrate, the amount or L-y-glutamyl-La-aminobutyrate formed was also estimated by using ~-a- [14C]aminobutyrate and L-glutamate as substrates. The amount of ~-y-glutamyl-~-a-['~c] aminobutyrate formed was approximately 93% of that determined by Pi release as indicated in Table 1, and the release of GSH-dependent inhibition by GSH conjugates was confirmed by independent test systems. The ATPase activity involved in the enzyme in the absence of L-glutamate and L-a-aminobutyrate was found to be negligible and less than 2% of the GC synthetase activity. The inhibitory effect of y-glutamyl-l-cysteine was not reduced the addition of GS-N2ph. Fig. 1 shows the effect of incubation time on inhibition by 10 mm GSH in the presence or absence of 1 pm GS-N2ph. Inhibition by GSH and increase in synthesis with the addition of GS-Nzph were linear with time. 133 Fig. 2 shows the restoration of the activity inhibited by 10 mm GSH as a function of various concentrations of GS-N2ph. The activity increased linearly in the range of pm GS-N,ph, and gradually at 1-50 pm. The maximal effect of added GS-N2ph was observed at 50 pm GS-N2ph and was 200% of the activity in the absence of GS-N2ph. Fig. 3 shows restoration of activity by GS-N2ph at various concentrations of GSH. The inhibition of enzyme activity by GSH was dependent on the concentration of GSH, with an apparent Ki of Fig. 2. Effect of various concentrations of GS-N2ph on the GC synthetase activity. The incubation mixture was the same as for Fig. 1 in the presence of 10 mm GSH and GS-Nzph ( pm). After incubation for 15 min at 37"C, Pi released was determined. Enzyme activity was expressed as a percentage of the activity in the absence of the conjugate. The insert is an expanded scale drawing to illustrate better the low-concentration range of GS-Nzph. This insert corresponds to the boxed area centered around the origin of the large graph Fig. 1. Effect of incubation time on the GC synthetase activity. The incubation mixture was composed of 0.1 M Tris/HCl, ph 8.2,lO mm L-glutamate, 10 mm L-a-aminobutyrate, 5 mm ATP, 20 mm MgCI2, 2 mm EDTA, 0.4% bovine serum albumin, 3 units of enzyme in a final volume of 0.5 ml in the presence of 10 mm GSH (O), or both 10mM GSH and 1 pm GS-N,ph (A);(0) control without added GSH or GS-Nzph. Enzyme activity was determined as described under Methods Fig. 3. Restoration of GC synthetase activity by GS-N2ph at various concentrations of GSH. The incubation mixture comprised the same components as in Fig. 1 in the presence or absence of GS-Nzph at various concentrations of GSH (0-10 mm). The mixture was incubated for 15 min at 37 C. (0) No GS-Nzph; (0) 1 pm GS- Nzph; (A) 5 PM GS-Npph
4 134 Fig. 4. Effect of GS-Nzph on the GC synthetase activity. (A) Effect of GS-N2ph at various concentrations of L-glutamate; (C) effect of GS- Nzph at various concentrations of L-a-aminobutyrate. (B, D) Double-reciprocal plots of the data of A, C. The incubation mixture was composed of 0.1 M Tris/HCl, ph 8.2, 10 mm ATP, 50 mm MgC12, 2 mm EDTA, 0.4% bovine serum albumin, 1 unit of enzyme and 10 mm GSH in a final volume of 0.5 ml, in (A) 10 mm L-a-aminobutyrate and L-glutamate as indicated; in (C) 10 mm L-glutamate and L-aaminobutyrate as indicated in the presence or absence of GS-N2ph. After incubation for 15 min at 37"C, enzyme activity was determined as described under Methods. (0) Control; (0) 10 mm GSH; (A) 10 mm GSH and 1 pm GS-NZph; (m) 10 mm GSH and 5 pm GS-Nzph Fig. 5. Chromatography of [3H]GSH and r3h]gsh S-conjugate. Erythrocytes were incubated with [3H]glycine in the presence (A) or absence (B) of CINzBz as outlined in Methods. The cells were lysed, treated with t-hydroperoxide to oxidize all of the GSH to GSSG, and dialyzed against water. The outer solution of dialysis was chromatographed on Dowex-I (formate form). The column was eluted with a 0-4 M formate gradient. The radioactivity that appeared before the gradient is from residual [3H]glycine 2.3 mm. Substantial restoration of activity was observed with the addition of 1 pm and 5 pm GS-Nzph. The effect of GS-N2ph was also studied as a function of various concentrations of L-glutamic acid (Fig. 4A) and L-aaminobutyric acid (Fig. 4C). The extent of release of inhibition by GSH by the addition of GS-N2ph appears to be dependent on the concentration of L-glutamate (Fig. 4A). The inhibition by GSH is competitive with respect to L-glutamate [20], and the relationship between GSH and GS-Nzph seems to be competitive, since the plot of l /v versus GSH concentration gives straight line (Fig. 3) and V is unaffected by the addition of GS-Nzph (Fig. 4B) [21]. On the other hand, the enzyme activity is also substantially increased with the increase of L-a-aminobutyrate (Fig. 4C). The inhibition by GSH is uncompetitive with respect to L-a-aminobutyrate (Fig. 4D). Effect of GSH S-conjugate on the biosynthesis of GSH in erythrocytes In order to determine the effect of formation of GS-Nzph on the biosynthesis of GSH in red cells, human red cells were incubated with [3H]glycine in the presence or absence of CINzBz, followed by the treatment with t-butylhydroperoxide to oxidize the synthesized GSH to GSSG. The GSSG and GS-Nzph formed in red cells were prepared using Dowex-1 chromatography. Fig. 5 shows the chromatographic isolation patterns of GSSG and GS-Nzph synthesized in erythrocytes using [3H]glycine. The level of labeled glycine incorporated into GSSG and GS-N2ph in the presence of ClN2Bz was 1.5- fold over the control levels.
5 135 DISCUSSION In human erythrocytes glutathione is present mainly in a reduced form at an approximate concentration of 2 mm and at a far lower concentration in the form of GSSG [19]. GSH synthesized in human erythrocytes is vital for the protection of the cells from oxidative stress and for the removal or circulating xenobiotics. The prgsent study on GSH S-conjugate reports that it increases the activity of GC synthetase, which is strongly inhibited by GSH. An apparent Ki of 2.3 mm with respect to GSH concentration for human erythrocyte GC synthetase was in fairly good agreement with that for the enzyme from rat kidney [lo]. It is conceivable that the mechanism by which the enzyme activity was stimulated by GSH S-conjugate is due to the release of inhibition by GSH. The results of the present study indicate that the effect of GS-N2ph on the activity of GC synthetase in the presence of GSH is dependent on the concentration of the S-conjugate ( pm). A linear increase in the enzyme activity is observed in the range of O pm GS-N2ph (Fig. 2). Addition of 1 pm GS-N2ph to the assay mixture in the presence of 10 mm GSH resulted in 45% increase in activity. Such a low concentration of GSH S-conjugate could stimulate the activity of GC synthetase at physiological concentrations of GSH. Addition of GS-N2ph to the assay mixture in the absence of GSH had no apparent effect on the activity of GC synthetase. Feedback inhibition of the enzyme by y-glutamyl- L-cysteine was not modulated by the addition of GS-N2ph. Richman and Meister previously reported that GSH competitively inhibits GC synthetase with respect to L-glutamate [20]. Maede et al. have recently found using canine erythrocytes with a hereditary high concentration of GSH that GC synthetase is activated by increasing the concentration of L-glutamate in the incubation media [22]. It has been suggested that GSH binds competitively to the glutamate site of the enzyme [20]. In this study, the releasing effect of GS-Nzph on inhibition of GC synthetase by GSH was kinetically characterized. The effect of GS-N2ph was substantial when L-glutamate concentration was increased. The relationship between GSH and GS-N2ph seemed to be competive, suggesting that GS-N2ph binds to the L-glutamate site of the enzyme. Under normal conditions GSH synthesis is mainly regulated by a feedback inhibition by GSH [23]. When erythrocytes are exposed to electrophilic compounds, intracellular GSH is depleted and GSH S-conjugates are accumulated [7]. The decrease in the level of GSH weakens the inhibitory effect on GC synthetase, and moreover GSH S-conjugate acts to increase the rate of GSH synthesis. On the other hand, ATPrequiring transport systems for GSSG [24, 251 and GSH S- conjugate [13] have been characterized. GSH S-conjugate in erythrocytes is eliminated via the same transport process used for high concentrations of GSSG [13], as observed similarly in rat liver [26]. In this study the effect of the formation of GSH S-conjugate on the biosynthesis of GSH was determined using human erythrocytes (Fig. 5). With the addition of CINzBz a 1 S-fold increase in the level of GSH was seen. It is suggested that GSH S-conjugate increased the GSH synthesis in erythrocytes. The idea that less transport of GSSG, due to a competitive inhibition by GSH S-conjugate, lead to higher levels of GSSG is not possible because GSH S-conjugate does not inhibit the transport at physiological concentrations of GSSG [13]. If removal of S-conjugates from the cells were not possible, exposure of erythrocytes to electrophiles at normal concentrations of GSH could lead to an accumulation of the Fig. 6. Metabolic interrelationship involved in glutathione synthesis in human erythrocytes. (1) GC synthetase; (2) glutathione synthetase; (3) GSH S-transferase; (4) glutathione reductase; (5) glutathione peroxidase. (Adapted from a diagram by Prins and Loos [27]) S-conjugates, which might in turn upset the GSH synthesis. Therefore the transport system of GSH S-conjugates may play an important role in glutathione metabolism in erythrocytes. Fig. 6 shows the interrelationship of the metabolic processes involved in human erythrocyte glutathione synthesis. The glutathone in erythrocytes is maintained at a fairly constant concentration by the regulation of GSH biosynthesis, especially at the level of GC synthetase, and by the active transport of GSSG or GSH S-conjugates out of the cells. The authors thank Dr George L. Dale and Dr S. Gasa for their valuable suggestions, Mrs Stephanie House and Miss Atsuko Shibuya for their technical assistance. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. Snoke, J. E. & Bloch, K. (1975) J. Biol. Chem. 213, Majerus, P. W., Brauner, M. J., Smith, M. B. & Minch, V. (1971) J. Clin. Invest. 50, Jacoby, W. B. (1978) Adv. Enzymol. 46, Chasseaud, L. F. (1977) Adv. Cancer Res. 29, Marcus, C. J., Habig, W. H. & Jacoby, W. B. (1978) Arch. Biochem. Biophys. 188, Harvey, J. W. & Beutler, E. (1982) Blood 60, Awasthi, Y. C., Charge, H. S., Dao, D. D., Parridge, C. A. & Srivastava, S. K. (1981) BloodS8, Bilzer, M., Kraus-Siegel, L., Schirmer, R. H., Akerboom, T. P., Sies, H. & Schulz, G. (1984) Eur. J. Biochern. 138, Beutler, E., West, C. & Blume, K. G. (1976) J. Lab. Clin. Med. 88, Sekura, R. & Meister, A. (1976) J. Biol. Chem. 2S2, Awasthi, Y. C., Dao, D. D. & Saneto, R. P. (1980) Biochem. J. 191,l Simons, P. C. & Vander Jagt, D. L. (1977) Anal. Biochem. 82, Kondo, T., Murao, M. & Taniguchi, N. (1982) Eur. J. Biochem. 125, Wahllander, A. & Sies, H. (1979) Eur. J. Biochem. 96,
6 Martin, J. B. & Doty, D. M. (1949) Anal. Chem. 21, Griffith, 0. W. & Meister, A. (1977) Proc. Natl Acad. Sci. USA 74, Orlowski, M. & Meister, A. (1971) J. Biol. Chem. 246, Lunn, G., Dale, G. L. & Beutler, E. (1979) Blood 54, Beutler, E. (1975) A Manual of Biochemical Methods, 2nd edn, Grune and Stratton, New York. 20. Richman, P. G. & Meister, A. (1975) J. Biol. Chem. 250, Keleti, T. (1967) J. Theor. Biol. 16, Maede, Y., Kasai, N. & Taniguchi, N. (1982) Blood 59, Meister, A. & Anderson, E. (1983) Annu. Rev. Biochem. 52, Kondo, T., Dale, G. L. & Beutler, E. (1980) Proc. Nutl Acad. Sci. USA 177, Kondo, T., Dale, G. L. & Beutler, E. (1981) Biochem. Biophys. Acta 645, Akerboom, T. P. M., Bilzer, M. & Sies, H. (1982) FEBS Lett. 140, Prins, H. K. & Loos, J. A. (1969) in Biochemical Methods in Red Cell Genetics (Yunis, J. J. ed.) pp , Academic Press, New York. T. Kondo and Y. Kawakami, First Department of Medicine, Hokkaido University School of Medicine, Kita-15, Nishi-7, Sapporo-shi, Hokkaido, Japan 060 N. Taniguchi, Biochemistry Laboratory, Cancer Institute, Hokkaido University School of Medicine, Kita-15, Nishi-7, Sapporo-shi, Hokkaido, Japan 060
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