Enzymes of glutamine metabolism in rat : subcellular localization of glutaminases in liver and kidney cortex and characterization of y-glutamyl transferring activities in rat kidney cortex

Abstract

Urinary excretion of ammonia facilitates excretion of strong acids and serves to conserve body stores of sodium. This ammonia is provided mainly by the renal hydrolysis of gltamine catalyzed by the glutaminase isoenzyrnes. The subcellular Iocalization of the isoenzymes of glutaminase has been studied in rat kidney, cortex and liver. Differential centrifugation and sucrose density gradient techniques demon-strated a mitochondrial localization for phosphate dependent glutaminase in both liver and kidney cortex. Fractionation of isolated mitochondria by digitonin and by sonication revealed that phosphate-dependent gliltaminase is located in the mitochondrial matrix compartment, a finding in agreement with its demonstrable latency. -- The highest specific activity of phosphate-independent glutaminase was found in the microsomal fraction of rat kidney cortex on differential centrifugation. This fraction was also enriched in NADPH-cytochrome c reductase (endoplasmic reticulum marker) ,5'-nucleotidase (plasma membrane marker), alkaline phosphatase, γ-glutamyltranspeptidase and maltase (brush border markers). Continuous sucrose density gradient studies on acrosomal fractions showed that phosphate-indeminase was located, in the brush border membranes, of rat kidney cortex. This enzyme is truly membranous as it could not be removed by sonication, salt treatment or pH alterations. -- Further studies on phosphate-independent glutaminase and γ-glutamyl transferring activities of rat kidney cortex showed that phosphate-independent glutaminase enzyme appears to be a single hydrolase which catalyses the hydrolosis of glutamine, glutathione, γ-gluamylhydroxamate and γ-gluamyl-p-nitroanilide. The enzyme activity was stimulated in the presence of maleate and competition between the substrates was observed. Phosphate-independent glutaminase activity was not attributed to the glutamine synthetase and it also appeared to be different from γ-glutamyltransferase activity. Phosphate-independent glutaminase and γ-glutamyltranspeptidase activities were lost to identical extents by various heat treatments, were removed identically from brush border membranes by papain treatment and were co-purified, on Sephadex G-100. Glutamine was shown to inhibit γ-glutamyltranspeptidase competitively with γ-gluamyl-p-nitroanilide. These studies suggests that phosphate-independent glutaminase and γ-glutamyltranspeptidase represent different catalytic actions of the same enzyme.