FEBS Letters | |
Purification of an NADPH‐dependent diaphorase from membrane of DMSO‐induced differentiated human promyelocytic leukemia HL‐60 cells | |
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
【 预 览 】
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