Inactivation of renal gamma-glutamyl transferase by 6-diazo-5-oxo-L-norleucylglycine, an inactive precursor of affinity-labeling reagent.
In vitro experiments showed that 6-diazo-5-oxo-L-norleucylglycine, a dipeptide analog of L-glutaminylglycine, inactivates gamma-glutamyl transferase bound to renal brush border membrane vesicles but does not inactivate the purified transferase. The rate of inactivation of the membrane-bound enzyme decreased markedly in the presence of dipeptides, such as L-leucylglycine and L-alanylglycine, or in the presence of o-phenanthroline, an inhibitor of renal peptidases. The presence of L-cysteinylglycine S-acetyldextran polymer (Mr 500,000), which does not permeate membranes, protected the membrane-bound transferase from inactivation by 6-diazo-5-oxo-L-norleucyglycine. This and other findings suggest that the norleucylglycine derivative was hydrolyzed by peptidase(s) bound to the outer surface of the brush border membranes and that the 6-diazo-5-oxo-L-norleucine thus released acts as an affinity-labeling reagent for the membrane-bound transferase. Similar effects were observed in vivo. Intravenous administration of 6-diazo-5-oxo-L-norleucylglycine to mice resulted in a marked decrease in renal transferase activity. Mice thus pretreated with 6-diazo-5-oxo-L-norleucylglycine, but not an untreated group, excreted significant amounts of S-carbamido[14C]methylglutathione in their urine within 30 min of intravenous administration of this compound. This finding suggests that the renal transferase was involved in the hydrolysis of the glutathione S-conjugate in the glomerular filtrate in vivo and that the administered 6-diazo-5-oxo-L-norleucylglycine underwent hydrolysis peptidase(s)-catalyzed to liberate 6-diazo-5-oxo-L-norleucine that reacted with the membrane-bound gamma-glutamyl transferase.
D000345:Affinity Labels / Q000378:metabolism / Q000494:pharmacology*; D000818:Animals; D001391:Azo Compounds / Q000494:pharmacology*; D003980:Diazooxonorleucine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology* / Q000652:urine; D007668:Kidney / Q000201:enzymology*; D007687:Kidney Tubules, Proximal / Q000201:enzymology; D007700:Kinetics; D008297:Male; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D051379:Mice; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors*
D000345:Affinity Labels; D001391:Azo Compounds; D008565:Membrane Proteins; D003980:Diazooxonorleucine; C030442:6-diazo-5-oxo-L-norleucylglycine; D005723:gamma-Glutamyltransferase