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.