title: Identification and characterization of lysosomal enzymes involved in the proteolysis of phenobarbital-inducible cytochrome P450.
abstract: We have studied lysosomal proteases capable of degrading a major form of cytochrome P450 (CYP2B1) which was purified from the liver microsomes of phenobarbital-treated rats. After incubation of CYP2B1 with extracts of triton-filled lysosomes (tritosomes), its proteolysis was measured by a quantitative immunoblot procedure. A CYP2B1 protein band with an apparent molecular mass of 53 kilodaltons (kDa) was degraded to 42-, 38-, and 29-kDa polypeptides after a short period of incubation. These proteolytic fragments disappeared on prolonged incubation. One milligram of tritosomal protein contained enough activity to hydrolyze approximately 67.3 micrograms CYP2B1 protein/min at pH 4.5. Approximately 85% of the hydrolyzing activity was localized in a soluble fraction of the tritosomes. The degradation of CYP2B1 was effectively inhibited by pepstatin A, an aspartic protease inhibitor, but not by phenylmethanesulfonyl fluoride (PMSF), o-phenanthroline and leupeptin. CYP2B1-hydrolyzing activity was coeluted with cathepsin D when the soluble fraction was chromatographed by means of Ultrogel Ac44 gel filtration. Cathepsin D, purified from rat livers, was able to degrade CYP2B1 at pH 4.5 which corresponds to the intralysosomal pH. These results indicate that cathepsin D is responsible for the major CYP2B1-hydrolyzing enzyme activity in lysosomes.
authors.affiliation: Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Hiroshima, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Tsuji
authors.forename: K
authors.initials: K
authors.lastname: Akasaki
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002402:Cathepsin D / Q000378:metabolism; D002850:Chromatography, Gel; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008247:Lysosomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013250:Steroid Hydroxylases / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D011480:Protease Inhibitors; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C025619:steroid 16-beta-hydroxylase; D010450:Endopeptidases; D002402:Cathepsin D; D010634:Phenobarbital