[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase.
[3H]Guanidinoethylmercaptosuccinic acid (GEMSA), a potent inhibitor of enkephalin convertase, binds to membrane and soluble fractions of tissue homogenates saturably and reversibly with a KD of 6 nM. Specific binding accounts for greater than 95% of total binding. The highest levels of [3H]GEMSA binding occur in the pituitary gland and the brain, with much lower levels in peripheral tissues. GEMSA, guanidinopropylsuccinic acid, 2-mercaptomethyl-3-guanidinothiopropionic acid, aminopropylmercaptosuccinic acid, [Leu] enkephalin-Arg, and [Met]enkephalin-Arg inhibit [3H] GEMSA binding to crude rat brain homogenates, to crude bovine pituitary homogenates, and to pure enkephalin convertase with equal potencies. Their Ki values against [3H]GEMSA binding are similar to their Ki values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the Vmax for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the Bmax (maximal number of binding sites) for [3H]GEMSA is about 2,000 min-1 in both pure enzyme preparations and crude tissue homogenates. [3H] GEMSA binding activity is found only in fractions containing enkephalin convertase during enzyme purification from bovine pituitary by L-arginine affinity chromatography. These data confirm that [3H]GEMSA binds only to enkephalin convertase in crude homogenates under our assay conditions. CoCl2 activates enzyme activity without altering the Ki of GEMSA against enzymatic hydrolysis and weakly inhibits [3H] GEMSA binding by increasing the KD.
D000818:Animals; D001921:Brain / Q000378:metabolism; D043423:Carboxypeptidase H; D002268:Carboxypeptidases / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D003619:Dansyl Compounds / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010902:Pituitary Gland / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013386:Succinates / Q000378:metabolism*; D014018:Tissue Distribution
D002413:Cations, Divalent; D003619:Dansyl Compounds; D009842:Oligopeptides; D011480:Protease Inhibitors; D013386:Succinates; C022014:guanidinoethylmercaptosuccinic acid; C043048:dansyl-phenylalanyl-leucyl-arginine; D002268:Carboxypeptidases; D043423:Carboxypeptidase H