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Membrane-Bound Oxidaseactivity Proc. Natl. Acad. Sci. USA Vol. 76, No. 6, pp. 2715- 2719, June 1979 Biochemistry Conversion of glutathione to glutathione disulfide by cell membrane-bound oxidase activity ("y-glutamyl transpeptidase/'y-glutamyl cycle/amino acids/kidney/jejunum) SURESH S. TATE, ELENA M. GRAU, AND ALTON MEISTER Department of Biochemistry, Cornell University Medical college, 1300 York Avenue, New York, New York 10021 Contributed by Alton Meister, April 2, 1979 ABSTRACT An apparently specific glutathione oxidase 12) on the metabolism of glutathione and of a glutathione activity is present in renal cortex, epididymal caput, jejunal conjugate by isolated rat kidney cells have confirmed these villus tip cells, choroid plexus, and retina (but not in liver). The findings and have also provided evidence for stepwise break- activity is membrane-bound and is localized on the luminal constituent amino surface of the brush border membranes of the kidney and je- down of glutathione disulfide to its acids, junum. The distribution and localization of the oxidase are reactions that are presumably catalyzed by the combined ac- similar to those of 'y-glutamyl transpeptidase, suggesting that tions of 'y-glutamyl transpeptidase and dipeptidase. These there is a significant relationship among the translocation of workers found little or no activity in studies with suspension of intracellular glutathione, the extracellular oxidation of gluta- liver cells, but they found that the reaction mediated by sIus- thione to glutathione disulfide, and the reactions of the y-glu- pensions of kidney cells was inhibited by cyanide or by re- tamyl cycle. Thus, both glutathione present in the blood plasma with and intracellular glutathione translocated to the cell surface are placement of the atmosphere nitrogen. accessible to oxidation and transpeptidation. Acceptor sub- In the present work we have found that there is an apparently strates of the transpeptidase (e.g., L amino acids) promote specific glutathione oxidase activity in kidney, epididymis, transpeptidation and decrease oxidation of glutathione. Con- jejunum, choroid plexus, and other tissues. * The activity is version of glutathione to glutathione disulfide is followed by membrane-bound and is associated with the brush border of utilization of the latter compound by 'y-glutamyl transpeptidase the kidney and jejunum. Its distribution in tissues and in tissue and dipeptidase. Although intracellular oxidation of glutathione to that of trans- to glutathione disulfide is readily reversed by the action of fractions follows a pattern similar 'y-glutamyl glutathione reductase, glutathione disulfide formed extracel- peptidase; however, the oxidase was not found in two lymphoid lularly cannot be reduced; instead, it undergoes hydrolytic and cell lines that have moderate and high levels of -y-glutamyl transpeptidation reactions leading to y-glutamyl amino acid transpeptidase activity. The distribution and localization of the and amino acid products which may be recovered by being oxidase suggest that there is a significant relationship among transported into the cell. the function of the -y-glutamyl cycle, the translocation of glu- oxidation of to glu- Recent studies on the metabolism of glutathione have provided tathione, and the extracellular glutathione new evidence that the reactions of the y-glutamyl cycle (1) tathione disulfide. occur in vivo (2-5) and that translocation of glutathione across EXPERIMENTAL renal cell membranes is a discrete step in the cycle (6, 7). The available data also indicate that intracellular glutathione is Materials. Glutathione, S-methylglutathione, L--y-gll- translocated out of other cells-e.g., liver (6, 8) and lymphoid tamyl-p-nitroanilide, glutathione reductase (yeast), papain, cells (7). Cells that have relatively little -y-glutamyl transpep- NADPH, and concanavalin A-Sepharose were obtained from tidase activity translocate glutathione into the blood plasma Sigma. L-Cysteinylglycine was prepared by incubating gluta- whereas, in renal cells, intracellular glutathione is translocated thione with purified rat kidney -y-glutamyl transpeptidase (14), to membrane-bound 'y-glutamyl transpeptidase. Normally, followed by chromatography of the reaction products on a little, if any, glutathione is found in the urine, and the blood column of Dowex 1-acetate. L-'y-Glutamyl-(o-carboxy)- plasma levels of glutathione are maintained at extremely low phenylhydrazide was synthesized in this laboratory by Owen levels. However, after administration of a potent 'y-glutamyl W. Griffith (6). transpeptidase inhibitor to mice, the amount of glutathione Methods. Tissue homogenates were made in 4 vol (wt/vol) excreted in the urine increased substantially and the blood of 10 mM sodium phosphate/20 mM Hepes/0. 15 M NaCl, pH plasma level of glutathione also increased appreciably; most 7.4 (designated here as Hepes/phosphate buffer). The subcel- of the glutathione found in the urine was in the form of gluta- lular fractionation of rat kidney to yield basal-lateral and brush thione disulfide (6). More than half of the glutathione of blood border membranes and determination of marker enzymes have plasma is also present in the disulfide form. These observations been described (15). y-Glutamyl transpeptidase was assayed are consistent with the function of a system or systems that in the presence of 1 mM L-y-glutamyl-p-nitroanilide, 20 mM mediate the oxidation of extracellular glutathione. Gly-Gly, and 0.1 M Tris-HCI (pH 8.0); its activity is expressed Some years ago, Elvehjem and collaborators (9, 10) found that as ,qmol of p-nitroaniline released per min (units). Glutathione mouse kidney homogenates catalyze oxygen-dependent con- oxidase activity was assayed in reaction mixtures (final volume, version of glutathione to glutathione disulfide and that this 1 ml) containing Hepes/phosphate buffer, 2 mM glutathione, reaction is inhibited by cyanide. These workers also found that and the enzyme. Incubation was carried out with vigorous kidney exhibits much higher glutathione oxidase activity than shaking on a reciprocating shaker at 370C. Samples (0.2 ml) does liver. Recent studies by Orrenius and his colleagues (11, were withdrawn and mixed with 0.02 ml of either 50% sulfo- salicylic acid or 2 M acetic acid. After deproteinization, aliquots The publication costs of this article were defraved in part by page were taken for free thiol determination with 5,5'-dithiobis(2- charge payment. This article must therefore be hereby marked ''ad- vertisement" in accordance with 18 U. S. C. §1734 solely to indicate * A preliminary account of some of these findings has appeared this fact. (13). 2715 Downloaded by guest on September 30, 2021 2716 Biochemistry: Tate et al. Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Activities of GSH oxidase and -y-glutamyl Of interest is the gradient of oxidase activity found in jejunal transpeptidase (GGTP) in rat tissues* epithelial cells; these data resemble those found earlier for Ratio: transpeptidase (19, 21) which exhibits highest activity in the GGTP, Oxidase, GGTP/ differentiated villus tip cells. Under the conditions used, no Tissue units/mg units/mg oxidase oxidase activity could be detected in the liver. In the studies described above, the glutathione oxidase ac- Kidney: tivity was in Cortex 2.68 0.20 13 found the particulate fractions of the homogenates, Medulla 2.15 0.15 14 suggesting that the enzyme is membrane-bound. Subcellular fractionation of rat kidney carried out essentially as described Epididymis: (15) showed about an 18-fold enrichment of the oxidase ac- Caput 0.64 0.056 11 tivity in the brush border membrane fraction (Table 2; P5B). Cauda 0.09 0.009 10 This fraction also contained similarly enriched transpeptidase and alkaline phosphatase activities; these enzymes are consid- Jejunal epithelial cells:t ered to be brush border markers. The ratio of transpeptidase Crypt cells 0.019 0.0021 9 activity to oxidase activity remained constant during the frac- Mid fractions 0.052 0.0072 7 tionation procedure. The cytosolic fraction (which contained Villus tip cells 0.075 0.0117 6 microsomes; 16,000 X g supernatant of the postnuclear fraction) contained 6-8% of the oxidase and transpeptidase activities of Liver 6.5 X 10-4 ND the homogenate. * ND, not detectable. For transpeptidase (GGTP), units are umol of Treatment of brush border vesicles with papain led to solu- p-nitroaniline formed per min in tissue homogenates (assayed in bilization of about 65 and 60% of the oxidase and transpeptidase presence of 1 mM L-y-glutamyl-p-nitroanilide and 20 mM Gly-Gly). activities, respectively, indicating that a major portion of these For oxidase, units are Asmol of GSH oxidized per min (decrease in activities is located on the outer or luminal surface of the cell free thiol). Both are shown per mg of protein. membrane. Previous studies have shown that transpeptidase Fractions of the jejunal epithelial cells were obtained by the mi- is located on the outer surface of kidney cells as well as of brush crometer planing technique of Imondi et al. (19). border vesicles (1, 15, 23). Consistent with this interpretation is the finding that both oxidase and transpeptidase activities nitrobenzoate) (16), for total glutathione (GSH + GSSG) de- were unaffected by treatment of brush border vesicles with termination with glutathione reductase by the method of Tietze detergents (e.g., Lubrol WX and sodium deoxycholate). (17), and for glutathione disulfide determination. Glutathione Data on the activities of transpeptidase and the oxidase in disulfide was determined by treating aliquots of the reaction bovine tissues are given in Table 3. The ratios of the activities mixture with 2-vinylpyridine to derivatize the glutathione in kidney cortex, choroid plexus, and retina varied from 8 to present prior to application of the Tietze procedure (7). The 13. The ratio for kidney medulla was about 4, and no oxidase glutathione oxidase activity is expressed either as the decrease activity was detected in cerebral cortex. Studies were also car- in free thiols (umol/min) or as ,mol of glutathione disulfide ried out on two established human lymphoid cell lines, RPMI formed per min (units).
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