J. Biol. Chem., Vol. 259, Issue 19, 11812-11817, 10, 1984
[3H]guanidinoethylmercaptosuccinic acid binding to tissue homogenates. Selective labeling of enkephalin convertase
SM Strittmatter, DR Lynch and SH Snyder
[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.
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