Purification, characterization, and amino acid composition of rabbit pulmonary bleomycin hydrolase.
Bleomycin (BLM) hydrolase, a protective enzyme that inactivates the antitumor antibiotic BLM, was purified (6000-fold) to homogeneity from rabbit lungs by DEAE-Sephacel, phenyl-Sepharose chromatography, BLM-Sepharose affinity chromatography, and Mono Q fast protein liquid chromatography. The enzyme had a molecular mass of 250,000 daltons as demonstrated by Superose gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) under native conditions. Sodium dodecyl sulfate-PAGE revealed a single band of 50,000 daltons, suggesting a pentameric structure. The Km and Vmax for BLM A2 were 1.3 mM and 5.9 mumol mg-1 h-1, respectively. BLM hydrolase activity was labile, had a half-life of 25 min at 56 degrees C, 10 h at 37 degrees C, and 5 days at 4 degrees C, and was stabilized by 2 mM dithiothreitol. The enzyme had a pH optimum of 7.0-7.5 and was inhibited by N-ethylmaleimide, leupeptin, puromycin, and divalent cations such as Cu2+, Cd2+, Zn2+, and Co2+ but was unaffected by chelating agents. On the basis of Mono P chromatofocusing chromatography, three isoforms of BLM hydrolase (apparent pI's of 5.3, 4.5, and 4.3) were present in rabbit pulmonary cytosol. The elution profiles of BLM hydrolase from phenyl-Sepharose and Mono P chromatofocusing indicated that this enzyme is hydrophobic and acidic. This was confirmed by amino acid composition analysis, which demonstrated that 48% of the total amino acids of bleomycin hydrolase were hydrophobic and 37% were acidic.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002413:Cations, Divalent; D002852:Chromatography, Ion Exchange / Q000379:methods; D003546:Cysteine Endopeptidases; D003600:Cytosol / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D011817:Rabbits
D000596:Amino Acids; D002413:Cations, Divalent; D006026:Glycoside Hydrolases; D003546:Cysteine Endopeptidases; C031262:bleomycin hydrolase