Immunochemical and catalytical characterization of the human liver NADPH-cytochrome P450 reductase.
1. The NADPH-cytochrome P450 reductases (EC 1.6.2.4) from human and rabbit liver have been purified to electrophoretic homogeneity. The human reductase had an apparent monomeric molecular weight of 77,500 and the rabbit enzyme of 76,500. 2. Both flavoproteins exhibited typical flavoprotein spectra and contained equimolar quantities of FAD and FMN. The two reductases were catalytically active in reducing cytochrome c, ferricyanide and dichlorophenolindophenol, and in supporting rabbit liver cytochrome P450 Form 4 metabolism of 2-acetylaminofluorene. 3. An antibody raised in the goat against the human enzyme formed a precipitin line with the human reductase in a double-diffusion assay, but did not react with the rabbit reductase. Similarly, an antibody raised in the goat against the rabbit reductase formed a precipitin line with the rabbit enzyme, but did not cross-react with the human reductase. 4. Both antibodies inhibited cytochrome c reduction by the two reductases suggesting some immunochemical recognition. 5. Immunochemical cross-reactivity was confirmed when both reductases were subjected to the more sensitive immunoblot technique using either anti-human or anti-rabbit reductase IgG. 6. The human and rabbit reductases are essentially similar in amino acid composition, except that the former has larger amounts of serine and glycine.
McManus
M E
ME
Department of Clinical Pharmacology, School of Medicine, Flinders University, Bedford Park, South Australia.
Huggett
A
A
Burgess
W
W
Robson
R
R
Birkett
D J
DJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D015153:Blotting, Western; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology / Q000276:immunology; D009251:NADPH-Ferrihemoprotein Reductase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013045:Species Specificity
D000596:Amino Acids; D009251:NADPH-Ferrihemoprotein Reductase