Purification of an NADPH-dependent diaphorase from membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells.
NADPH diaphorase activity was found in membrane of DMSO-induced differentiated human promyelocytic leukemia HL-60 cells. This membrane-bound diaphorase activity increased dramatically during differentiation of HL-60 cells. A dye reductase was extracted from membrane of DMSO-induced differentiated HL-60 cells with n-octyl glucoside and sodium cholate in the presence of several protease inhibitors such as PMSF, DIFP, TLCK, antipain, chymostatin, leupeptin, pepstatin A and trypsin inhibitor. The NADPH diaphorase was highly purified by two-stage sequential column chromatographies. The purified enzyme, showing both SOD-insensitive cytochrome c and NBT reductase activities, migrated with an apparent molecular mass of 77 kDa on SDS-PAGE. When the purification of this diaphorase was carried out in the presence of only three protease inhibitors, PMSF, DIFP and TLCK, a partially proteolyzed form of the diaphorase with a molecular mass of 68 kDa was prepared. The proteolyzed diaphorase exhibited only an NADPH-dependent cytochrome c reductase. The NADPH diaphorase gave a positive cross-reaction to polyclonal antibodies raised against microsomal NADPH-cytochrome P450 reductase from rabbit liver.
Otsuka-Murakami
H
H
Department of Biochemistry, Aichi Medical University, Japan.
Nisimoto
Y
Y
D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D008058:Dihydrolipoamide Dehydrogenase / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006098:Granulocytes / Q000166:cytology / Q000201:enzymology; D006801:Humans; D015473:Leukemia, Promyelocytic, Acute; D008970:Molecular Weight; D014407:Tumor Cells, Cultured
D008058:Dihydrolipoamide Dehydrogenase; D004121:Dimethyl Sulfoxide