[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 K(D) 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 K(i) values against [³H]GEMSA binding are similar to their K(i) values against enkephalin convertase activity. EDTA and 1,10-phenanthroline markedly inhibit both binding and enzymatic activity. The ratio of the V(max) for 5-dimethylaminonaphthalene-1-sulfonyl-Phe-Leu-Arg to the B(max)(maximal number of binding sites) for [³H]GEMSA is about 20,000 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 K(i) of GEMSA against enzymatic hydrolysis and weakly inhibits [³H]GEMSA binding by increasing the K(D).