Renal glutamine utilization: glycylglycine stimulation of γ-glutamyltransferase

Abstract

Glycylglycine stimulation of renal glutamine utilization was studied on the homogenate, subcellular and purified enzyme level. The results clearly establish the existence of two glutamine utilizing pathways, the mitochondrial dependent L-glutamine amidohydrolase (PDG) and a second, extramitochondrial pathway. In contrast to the mitochondrial pathway which produces stoichiometric amounts of ammonia and glutamate, this second pathway hydrolyzes glutamine to produce ammonia and transfers the γ-glutamyl moiety, producing γ-glutamyl peptides. In the crude systems, containing cyclotransferase, the γ-glutamyl moiety appears mainly as 5-oxoproline; however, in the enzyme preparation, purified 112-fold, γ-glutamyl peptides (transpeptidation) and a small amount of glutamate (hydrolysis) appear. D-Glutamine was also hydrolyzed, in contrast to the stereospecific PDG, but at less than one-half the rate of the L-isomer. The molecular weight of this extramitochondrial D- and L-glutamine utilizing enzyme was estimated by gel filtration on a Sephadex G-200 column and found to be ≈70 000. Based on product formation, molecular weight estimation and copurification with the activity responsible for p-nitroanilide release from γ-glutamyl-p-nitroanilide, we conclude that this reaction is catalyzed by γ-glutamyltranspeptidase. Glycylglycine stimulated this enzyme to produce more ammonia while decreasing the appearance of glutamate; in contrast, the mitochondrial glutaminase was unaffected by glycylglycine. This extramitochondrial glutamine utilizing pathway can make a significant contribution to in vivo renal ammoniagenesis.