Affinity labeling of rat-kidney gamma-glutamyl transpeptidase.
The reaction of gamma-glutamyl transpeptidase from rat kidney with a glutamine analog, 6-diazo-5-oxo-L-norleucine, resulted in irreversible inactivation of the enzyme. The concentration of this reagent giving a half-maximum rate of inactivation was 6 mMat pH 7.5. The inactivation was prevented by the presence of reduced glutathione in a competitive fashion, which indicates the active-site-directed nature of this reagent. The rate of inactivation was greatly accelerated in the presence of maleate, which is known to enhance the glutaminase activity of this enzyme. The presence of maleate increased the maximum velocity of the inactivation, but did not affect the affinity of the enzyme for 6-diazo-5-oxo-L-norleucine. Inactivation of the enzyme with 6-diazo-5-oxo-L-[6=14C]norleucine as well as with 6-diazo-5-oxo-L[1,2,3,4,5-14C]norleucine resulted in a stoichiometric incorporation of radioactivity into the enzyme protein via covalent linkage. The amount of radioactivity incorporated was 1 mol 14C label/248000 g enzyme protein. A native enzyme preparation showing a single protein band on polyacrylamide gel electrophoresis gave four distinct bands upon sodium dodecylsulfate/polyacrylamide gel electrophoresis. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis of the 14C-labeled enzyme, only the band moving the fastest towards the anode was found to contain radioactivity. This finding indicates that this protein band represents the catalytic component of the enzyme.
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D005973:Glutamine / Q000031:analogs & derivatives*; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008298:Maleates / Q000494:pharmacology; D009646:Norleucine / Q000031:analogs & derivatives; D011485:Protein Binding; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D008298:Maleates; D005973:Glutamine; D009646:Norleucine; D005723:gamma-Glutamyltransferase