PURIFICATION AND CHARACTERIZATION OF BLEOMYCIN HYDROLASE, WHICH REPRESENTS A NEW FAMILY OF CYSTEINE PROTEASES, FROM RAT SKIN

Bleomycin (BLM) hydrolase, which hydrolyzes the carboxyamide bond in t he beta-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homo geneity in five steps in the presence of 2-mercaptoethanol: 45-55% amm onium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The n ative enzyme had a molecular mass of 280 kDa according to gel filtrati on. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, in dicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2-terminus was determined to be acetyl-Me t-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the ami no acid sequence database. The optimum pH of the enzyme was 7.5 with p epleomycin (PLM). The K-m and V-max values were 2.1 mM and 6.8 mu mol . mg(-1). h(-1) for PLM, and 1.8 mM and 7.2 mu mol . mg(-1). h(-1) for BLM-A(2), respectively. The enzyme activity was inhibited by iodoacet ic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pC MB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. Howe ver, cystatins A and C did not inhibit the activity, BLM hydrolase exh ibited broad aminopeptidase substrate specificity towards aminoacyl-be ta-naphthylamides such as basic, neutral, and hydrophobic amino acid r esidues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blott ing and immunohistochemical analyses showed that BLM hydrolase is ubiq uitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the m etabolism of antibiotics.