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 the beta-amino-alanine moiety, was purified from newborn rat skin. The enzyme was purified 2,500-fold over the crude extract to apparent homogeneity in five steps in the presence of 2-mercaptoethanol: 45-55% ammonium sulfate fractionation, followed by chromatographies on Sephacryl S-200, DEAE-cellulofine, Phe-Superose, and Mono Q ion-exchange. The native enzyme had a molecular mass of 280 kDa according to gel filtration. The subunit molecular mass was estimated as 48 kDa by SDS-PAGE, indicating that the enzyme was comprised of six identical subunits. The amino acid sequence of its NH2-terminus was determined to be acetyl-Met-Asn-Asn-Ala-Gly-Leu-Asn-Ser-Glu-Lys-, which was not found in the amino acid sequence database. The optimum pH of the enzyme was 7.5 with pepleomycin (PLM). The Km and Vmax values were 2.1 mM and 6.8 mu mol center dot mg-1 center dot h-1 for PLM, and 1.8 mM and 7.2 mu mol center dot mg-1 center dot h-1 for BLM-A2, respectively. The enzyme activity was inhibited by iodoacetic acid, N-ethylmaleinimide (NEM), and p-chloromercuribenzoic acid (pCMB) as well as divalent cations such as Cu2+, Cd2+, Hg2+, and Zn2+. It was effectively inhibited by a cysteine protease inhibitor E-64. However, cystatins A and C did not inhibit the activity. BLM hydrolase exhibited broad aminopeptidase substrate specificity towards aminoacyl-beta-naphthylamides such as basic, neutral, and hydrophobic amino acid residues, as well as acidic residues. These results indicated that BLM hydrolase represents a new family of cysteine proteases. Western blotting and immunohistochemical analyses showed that BLM hydrolase is ubiquitous in various rat tissues but at low levels in lung and adult skin tissues, suggesting that this enzyme plays an important role in the metabolism of antibiotics.
Takeda
A
A
Department of Clinical Pathology, Showa University Fujigaoka Hospital, Yokohama.
Higuchi
D
D
Yamamoto
T
T
Nakamura
Y
Y
Masuda
Y
Y
Hirabayashi
T
T
Nakaya
K
K
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D003546:Cysteine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D012867:Skin / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity; D014018:Tissue Distribution
D000596:Amino Acids; D008670:Metals; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase