Purification and characterization of liver cytochrome P-446 isolated from protein energy malnourished rats.
A liver cytochrome P-450 isozyme has been purified to homogeneity from protein-energy malnourished rats induced with beta-naphthoflavone (beta-NF). The purification steps included chromatography on DEAE-Sephadex-A-25, DEAE-cellulose (DE-53), hydroxylapatite (HA) and carboxymethyl-sephadex (CM) columns. The reduced carbon monoxide difference and absolute spectra showed a Soret peak at 446.5 nm. The wavelength maxima for the oxidized and reduced spectra were at 416 and 408 nm, respectively. Cytochrome P-446 appears to have a predominantly low spin ferric iron, migrates as a single band of molecular weight 56,000 in sodium dodecyl sulfate polyacrylamide gels and has a specific content of 14 nmol/mg of protein. P-446 oxidized various substrates at different rates in a reconstituted system with NADPH-cytochrome P-450 reductase and dilauroyl-phosphatidylcholine. In this system turnover rates for benzo[alpha]pyrene, testosterone and benzphetamine oxidation were: 81.10; 1.85 and 1.42 nmoles product/min/nmol P-446 respectively. While NH2 terminal amino acid sequence analysis of 18 of the first 20 residues suggests that the cytochrome P-446 isolated from malnourished rats is identical with form c, the catalytic activities suggest that this isozyme may be a more effective or efficient catalyst for some substrates.
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D001589:Benzphetamine / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification*; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011502:Protein-Energy Malnutrition / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D007527:Isoenzymes; D001589:Benzphetamine; D001564:Benzo(a)pyrene; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System