Purification and characterization of NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes.
NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2',5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80,000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demethylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymorphonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.
D000818:Animals; D001589:Benzphetamine / Q000378:metabolism; D002845:Chromatography; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006900:Hydroxylation; D007163:Immunosorbent Techniques; D008862:Microsomes, Liver / Q000201:enzymology; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000097:blood*; D009504:Neutrophils / Q000201:enzymology*; D010713:Phosphatidylcholines / Q000494:pharmacology; D011817:Rabbits; D013552:Swine
D010713:Phosphatidylcholines; D001589:Benzphetamine; D005182:Flavin-Adenine Dinucleotide; C015153:1,2-dilauroylphosphatidylcholine; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase