Abstract
We have cloned the BLH1 gene of the yeast Saccharomyces cerevisiae coding for a peptidase with significant homology to rabbit bleomycin hydrolase. Bleomycin is a glycopeptide antibiotic used for the treatment of human cancers. The antitumor activity of the drug is limited by its metabolic inactivation caused by bleomycin hydrolase, a member of the cysteine protease family. The open reading frame of BLH1 consists of 1,449 base pairs encoding a 55.4-kDa protein consistent with the molecular mass found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The consensus sequence within the active site region of cysteine proteases is conserved in the yeast enzyme. Biochemical studies on the purified enzyme confirm its classification as a thiol protease. The nonvacuolar enzyme has a molecular mass of 220 kDa, suggesting a homotetrameric structure. It exhibits an aminopeptidase activity with broad substrate specificity. Biochemical and genetic linkage data give evidence that the BLH1 and the LAP3 (Trumbly, R. J., and Bradley, G. (1983) J. Bacteriol. 156, 36-48) encoded aminopeptidases are identical. Deletion of the BLH1 gene is not lethal under normal growth conditions. However, blh1 mutants show hypersensitivity to bleomycin, indicating that bleomycin hydrolase is able to inactivate bleomycin in vivo and to protect cells from bleomycin-induced toxicity.
Highlights
We have cloned the BLHl gene of the yeast Saccharomyces cerevisiaecoding for a peptidase with significant homology to rabbit bleomycin hydrolase
We report on the isolation and sequence determination of the BLHl gene of the yeast Saccharomyces cerevisiae coding for a new aminopeptidase that is the equivalent of the mam
Purified bleomycin hydrolase or molecular mass standards were applied on the Superose 6 column and eluted under the conditions as described in purification step 4
Summary
Hydrolase malian bleomycin hydrolase (Sebti et al, 1989). We further present the purification and characterization of the enzyme and report on theblhl null mutation in cell physiology. To clone the BLHl gene the pre mutant strain C13-9C, which is devoid of Cbz-Leu-Leu-Glu-pNA hydrolyzing activity (Hilt et al, 1993),was transformed with a yeast genomic library based on the 2pm URA3 shuttle vector pFLl (Chevallier et al, 1980). The purification was followed by testing all fractions for bleomycin hydrolase activity using Glu-DNA as substrate. Protein was eluted from the column with an increasing salt gradient Purified bleomycin hydrolase or molecular mass standards were applied on the Superose 6 column and eluted under the conditions as described in purification step 4.
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