GAMMA-Glutamyl transpeptidase of sheep-kidney cortex. Isolation, catalytic properties and dissociation into two polypeptide chains.
Gamma-Glutamyl transpeptidase was isolated from sheep kidney cortex as an apparently homogeneous, highly active protein. At optimal pH and in the absence of acceptors, the enzyme catalyzes the release of about 510 mumol of p-nitroaniline per mg protein per min from the model substrate L-gamma-glutamyl-p-nitroanilide. Polyacrylamide gel electrophoresis in a sodium dodecylsulfate buffer system showed the presence of a large (Mr approximately 65000) and a small (Mr approximately 27000) polypeptide chain. Dissociation into two polypeptide chains was also achieved in 8 M urea. Amidination with dimethylsuberimidate produced a crosslinked protein of molecular weight approximately 90000. In the course of this work a convenient procedure was developed for the determination of gamma-glutamyl transpeptidase activity using L[glycine-2-3H]glutathione as the substrate. In this procedure the release of cysteinyl-[2-3H]glycine from glutathione is followed, after separation of the radioactive di-peptide from unreacted glutathione on a small Dowex-1 acetate column. The reactions with gamma-glutamyl-p-nitroanilide and glutathione are both strongly activated by several metal ions (Ca2+, Mg2+, Na+ and K+) and by a number of amino acids and peptide acceptors. The products of the reaction with glutathione were identified as cysteinylglycine, gamma-glutamylglutathione and glutamate. The formation of these products is consistent with the function of gamma-glutamyl transpeptidase in both the gamma-glutamyl transfer reaction and in the hydrolysis of the gamma-glutamyl bond. The activating effect of metal ions in the reaction with glutathione was shown to be dependent on the acceleration of the transfer reaction; the rate of hydrolysis of the gamma-glutamyl bond remaining unchanged.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D008970:Molecular Weight; D011188:Potassium / Q000494:pharmacology; D012756:Sheep; D012964:Sodium / Q000494:pharmacology; D013329:Structure-Activity Relationship; D005723:gamma-Glutamyltransferase / Q000302:isolation & purification / Q000378:metabolism*
D046911:Macromolecular Substances; D012964:Sodium; D005723:gamma-Glutamyltransferase; D008274:Magnesium; D011188:Potassium; D002118:Calcium