Characterization of superoxide dismutase-insensitive cytochrome c reductase activity in HL-60 cytosol as NADPH-cytochrome P450 reductase.
A flavoprotein dehydrogenase assayed for the activity of electron transfer from NADPH to cytochrome c was highly purified from the cytosolic fraction of differentiated human promyelocytic leukemia HL-60 cells. The purified enzyme had an apparent molecular mass of 68 kDa by sodium dodecyl sulfate gel electrophoresis and an equimolar amounts of flavin mononucleotide and flavin-adenine dinucleotide. The purification factor of the enzyme with respect to the cytosolic fraction was close to 1100 and the recovery of activity was approximately 18%. Reduction of cytochrome c by NADPH indicated Michaelis-Menten kinetics with a Km value of 1.50 microM for NADPH. When cytochrome c was the varied substrate, a Km value of 4.10 microM was obtained. NADH was not an effective electron donor for cytochrome c reduction and NADPH-dependent reduction of nitroblue tetrazolium was negligibly small. The purified enzyme alone did not exhibit superoxide production, and NADPH oxidase activity was not markedly stimulated upon incubation of the reductase with cytochrome b558 purified from porcine neutrophils. The purified flavoprotein gave a positive cross-reactivity to polyclonal antibodies raised to microsomal NADPH-cytochrome P450 reductase, indicating structural homology between these enzymes. The catalytic properties of the purified NADPH-cytochrome c reductase have similarities to those of liver NADPH-cytochrome P450 reductase.
Nisimoto
Y
Y
Department of Biochemistry, Aichi Medical University, Japan.
Otsuka-Murakami
H
H
Iwata
S
S
Isogai
Y
Y
Iizuka
T
T
D000911:Antibodies, Monoclonal; D003429:Cross Reactions; D003573:Cytochrome b Group / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006801:Humans; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008862:Microsomes, Liver / Q000201:enzymology; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases; D009251:NADPH-Ferrihemoprotein Reductase / Q000187:drug effects / Q000276:immunology / Q000502:physiology*; D009504:Neutrophils / Q000201:enzymology; D013482:Superoxide Dismutase / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D003573:Cytochrome b Group; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D005486:Flavin Mononucleotide; C036888:cytochrome b558; D013482:Superoxide Dismutase; D009251:NADPH-Ferrihemoprotein Reductase; D019255:NADPH Oxidases