Gamma-Glutamyl transferase: studies of normal and cystinotic human leukocytes, rabbit neutrophiles, and rat liver.

Abstract

Summary: Evidence has been obtained in three different cell types, by a combination of biochemical and histologic approaches, that some y-glutamyl transferase (EC 2.3.2.2) activity is associated with lysosomes. The distribution of y-glutamyl transferase in subcellular fractions of human leukocytes and its enrichment in a postnuclear granule fraction were similar to the corresponding findings for lysosomal marker enzymes. Isopycnic centrifugation of the postnuclear fraction showed that although the bulk of the y-glutamyl transferase activity was associated with nonlysosomal particles containing alkaline phosphatase, a clearly separated fraction (approximately 20%) comigrated with lysosomal marker enzymes. L-cystine was the most potent of the amino acids tested as acceptors of the γ-glutamyl moiety of L-γ-glutamyl-P-nitroanilide. Apparent Km for L-cystine was 1.3 mM compared to 8.7 mM for L-glutamine, the second best amino acid acceptor. Optimum pH for the transferase was 9.0; there was no activity below pH 6. Further evidence that leukocyte γ-glutamyl transpeptidase includes a lysosomal component was obtained by demonstrating histochemically that the transpeptidase appears in cytoplasmic granules in rabbit neutrophile bone marrow precursors at a time when the synthesis of azurophile (lysosomal) granules is predominant. In addition, relative specific activity of the enzyme in the liver lysosomal fraction of rats preinjected with Triton WR-1339 was approximately 11. A comparison of its distribution with that of marker enzymes for the different subcellular fractions suggested that the lysosomal γ-glutamyl transferase activity is not accounted for by contamination with plasma membranes, endoplasmic retic-ulum or mitochondria. The possible mediation of amino acid transport by the transferase, for which cystine is an excellent γ-glutamyl acceptor, and a lysosomal location of some of the enzyme activity suggested that its deficiency might explain the intralysosomal storage of cystine in cystinosis. In these studies, however, no evidence was found for an abnormality of the enzyme in cystinotic leukocytes. Speculation: The suggestion that γ-glutamyl transpeptidase is a significant mediator of mammalian amino acid transport, and particularly the transport of cystine, has found little support in our present or previous investigations. The transpeptidase clearly catabolizes glutathione and has a complex intracellular distribution including an association with lysosomal particles, but its further functions, if any, and the significance of its subcellular localization remain to be defined.