A NOVEL ATPase OF ERYTHROYCTE MEMBRANES IN RELATION TO TRANSPORT OF GLUTATHIONE AND GLUTATHIONE CONJUGATES.

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We have purified a novel ATPase in human erythroyctes which is stimulated by oxidized form of glutathione(GSSG). Purification of the enzymes were performed to apparent homogeneity involving procedures of affinity chromatography on S-hexylglutathione-Sepharose 6B. Further characterization of the enzyme was performed using enzymatic, biochemical, physiological and immunological methods. The enzyme has two independent isozymes(type I and type II) which are indistinguishable by gel filtration, with an apparent molecular weight of 150,000. However they showed a different affinity for GSSG and ATP. We prepared polyclonal antibody using rabbits specific for each isozyme. These enzymes were immunologically cross reactive. The enzyme reconstituted into phospholipid vesicles showed both GSSG- stimulated ATPase activity and active GSSG transport activity. The GSSG-ATpase activity and the GSSG transport activity of type I isozyme were 25 nmol/mg protein/min, and 25 nmol pi released/mg protein/min, and those of type II isozyme were 248 nmol/mg protein/min and 133 nmol pi released/mg protein/min, respectively. These results indicate that these enzymes function in the active transport of GSSG and glutathione conjugates.