Carboxamidopeptidase: purification and characterization of a neurohypophyseal hormone inactivating peptidase from toad skin

Abstract

Carboxamidopeptidase, an enzyme which inactivates neurohypophyseal hormones, was purified 3800-fold in an overall yield of 22% from toad skin, a neurohypophyseal hormone target organ, by (NH4)2SO4 fractionation, DEAE-Sephadex chromatography, and affinity chromatography on immobilized p-aminobenzamidine and concanavalin A-agarose. The purified enzyme is capable of inactivating both [8-argnine]vasopressin (AVP) and oxytocin by hydrolyzing the Arg8.sbd.Gly9.sbd.NH2 and the Leu8.sbd.Gly9.sbd.NH2 bonds, respectively, and can hydrolyze the ester substrates, benzoyl-L-arginine ethyl ester (BzArgOEt) and acetyl-L-tyrosine ethyl ester, suggesting that the enzyme has both trypsin-like and chymotrypsin-like activities. Carboxamidopeptidase is maximally active at pH 7.5-8.5 for AVP and BzArgOEt and pH 7.0 for oxytocin. Carboxamidopeptidase is inhibited by ovoinhibitor, ovomucoid, Trasylol, lima bean trypsin inhibitor, concanavalin A, antipain, leupeptin, chymostatin, elastatinal, p-nitrophenyl p-guanidinobenzoate and 4-methylumbelliferyl p-guanidinobenzoate but not by soybean trypsin inhibitor, .alpha.1-antitrypsin, hirudin, pepstatin, bestatin, phosphoramidon or cysteine. The enzyme is also inhibited by the serine protease inhibitor, diisopropyl phosphofluoridate (i-Pr2PF), and by the chloromethyl ketone derivatives of tosyllysine, tosylphenylalanine, and (benzyloxycarbonyl)phenylalanine, as well as by the sulfhydryl group reagent, p-(chloromercuri)benzoate (PCMB). Inhibition by PCMB is reversed by cysteine. The MW determined by gel filtration in the presence of 1 M NaCl is approximately 100,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of 2 identical subunits of 48,000 daltons. Each subunit consists of a H chain (28,000 daltons) and a L chain (19,000 daltons) joined by a disulfide bond(s). Labeling experiments using [3H]-i-Pr2PF showed that the enzyme active site is located in the H chain.