Acetazolamide inhibition of renal gamma-glutamyl transpeptidase

The effect of acetazolamide (AZ) on renal gamma-glutamyl transpeptidase (EC 2.3.2.2) activity (gamma-GT) was studied with the purified enzyme, subcellular fractions, and in the isolated functioning kidney. Activity of gamma-GT was assessed using either one of two gamma-glutamyl donors, gamma-glutamyl-p-nitroanilide (gamma GpNA) or glutamine, and either the gamma-glutamyl acceptor glycylglycine (Gly-Gly) or methionine (Met). With the microsomal enzyme and beta-GpNA, AZ was shown to inhibit p-nitroaniline (p-NA) formation; however, gamma-GpNA Km remained unchanged (1.8 mM), while the Vmax was reduced significantly, 333 vs 200 mumoles . min-1 . mg-1. Adding Gly-Gly removed AZ inhibition, while AZ elevated the apparent Km from Gly-Gly from 16 to 48; AZ inhibition of gamma-GT activity resulted in a decrease in gamma-glutamyl-Gly-Gly formation consistent with interaction at the gamma-glutamyl acceptor site. With glutamine as the beta-glutamyl donor, AZ reduced NH3 and apparent gamma-glutamylmethionine formation in the purified enzyme in agreement with inhibition at the acceptor site. In the functioning kidney, perfused with 10(-3)M L- or D-glutamine, AZ (10(-3)M) markedly reduced NH3 formation and increased glutamine excretion, results consistent with AZ inhibition of the in situ gamma-GT.