Purification of an NADPH‐dependent diaphorase from membrane of DMSO‐induced differentiated human promyelocytic leukemia HL‐60 cells

FEBS Letters

Nisimoto, Yukio¹  Otsuka-Murakami, Hidetsugu¹ 

[1] Department of Biochemistry, Aichi Medical University, Nagakute, Aichi 480-11, Japan

关键词: NADPH-dependent diaphorase;    Cytochrome P-450 reductase;    HL-60 cell;    Neutrophil;    P450 reductase;    microsomal NADPH-cytochrome P-450 reductase from rabbit liver;    DMSO;    dimethyl sulfoxide;    DIFP;    diisopropyl fluorophosphate;    NBT;    Nitroblue tetrazolium;    OG;    n-octyl β-d-glucopyranoside;    PAGE;    polyacrylamide gel-electrophoresis;    PMA;    phorbol 12-myristate 13-acetate;    PMSF;    phenylmethylsulfonyl fluoride;    SOD;    superoxide dismutase;    TLCK;    Nα-p-tosyl-l-lysin chloromethyl ketone;    NADPH diaphorase;    NADPH-dependent diaphorase;   

DOI  :  10.1016/0014-5793(95)00183-A

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown