title: Purification and characterization of a doxorubicin-inhibited NADH-quinone (NADH-ferricyanide) reductase from rat liver plasma membranes.
abstract: Plasma membrane-associated redox systems play important roles in regulation of cell growth, internal pH, signal transduction, apoptosis, and defense against pathogens. Stimulation of cell growth and stimulation of the redox system of plasma membranes are correlated. When cell growth is inhibited by antitumor agents such as doxorubicin, capsaicin, and antitumor sulfonylureas, redox activities of the plasma membrane also are inhibited. A doxorubicin-inhibited NADH-quinone reductase was characterized and purified from plasma membranes of rat liver. First, an NADH-cytochrome b(5) reductase, which was doxorubicin-insensitive, was removed from the plasma membranes by the lysosomal protease, cathepsin D. After removal of the NADH-cytochrome b(5) reductase, the plasma membranes retained a doxorubicin-inhibited NADH-quinone reductase activity. The enzyme, with an apparent molecular mass of 57 kDa, was purified 200-fold over the cathepsin D-treated plasma membranes. The purified enzyme had also an NADH-coenzyme Q(0) reductase (NADH: external acceptor (quinone) reductase; EC 1.6.5.) activity. Partial amino acid sequence of the enzyme showed that it was unique with no sequence homology to any known protein. Antibody against the enzyme (peptide sequence) was produced and affinity-purified. The purified antibody immunoprecipitated both the NADH-ferricyanide reductase activity and NADH-coenzyme Q(0) reductase activity of plasma membranes and cross-reacted with human chronic myelogenous leukemia K562 cells and doxorubicin-resistant human chronic myelogenous leukemia K562R cells. Localization by fluorescence microscopy showed that the reaction was with the external surface of the plasma membranes. The doxorubicin-inhibited NADH-quinone reductase may provide a target for the anthracycline antitumor agents and a candidate ferricyanide reductase for plasma membrane electron transport.
authors.affiliation: Department of Medicinal Chemistry, Purdue University, West Lafayette, Indiana 47907, USA.
authors.forename: Chinpal
authors.initials: C
authors.lastname: Kim
authors.forename: Frederick L
authors.initials: FL
authors.lastname: Crane
authors.forename: W Page
authors.initials: WP
authors.lastname: Faulk
authors.forename: D James
authors.initials: DJ
authors.lastname: Morré
mesh_terms: D000818:Animals; D000838:Anions; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D015153:Blotting, Western; D002402:Cathepsin D / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000494:pharmacology*; D004579:Electron Transport; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D006801:Humans; D020014:K562 Cells; D008099:Liver / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification*; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D000838:Anions; D000903:Antibiotics, Antineoplastic; D009243:NAD; D004317:Doxorubicin; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D002402:Cathepsin D