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Purification of an NADPH‐dependent diaphorase from membrane of DMSO‐induced differentiated human promyelocytic leukemia HL‐60 cells
Author(s) -
Otsuka-Murakami Hidetsugu,
Nisimoto Yukio
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00183-a
Subject(s) - acute promyelocytic leukemia , chemistry , leukemia , microbiology and biotechnology , biochemistry , biology , immunology , gene , retinoic acid
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.