Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography.
14 microsomal cytochromes P-450 were purified from the liver of untreated and phenobarbital- or 3-methylcholanthrene-treated male rats. Following solubilization of microsomes with sodium cholate, poly(ethylene glycol) fractionation and aminohexyl-Sepharose 4B chromatography, cytochromes P-450 were purified by high-performance liquid chromatography (HPLC), using a preparative DEAE-anion-exchange column. The pass-through fraction was further purified by HPLC using a cation-exchange column. Other fractions eluted on preparative DEAE-HPLC were further applied onto an HPLC using a DEAE-column. Five kinds (P-450UT-2-6), four kinds (P-450PB-1,2,4 and 5) and five kinds (P-450MC-1-5) of cytochromes P-450 were purified from untreated rats or rats treated with phenobarbital or 3-methylcholanthrene, respectively. HPLC profiles of tryptic peptides of cytochromes P-450UT-2 and P-450MC-2 were identical and the other profiles obtained from seven purified cytochromes P-450 were distinct from each other. Amino-terminal sequences of eight forms of cytochrome P-450 (UT-2, UT-5, PB-1, PB-2, PB-4, PB-5, MC-1 and MC-5) were distinct except for cytochromes P-450PB-4 and P-450PB-5.
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D009251:NADPH-Ferrihemoprotein Reductase / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; D014357:Trypsin; D010634:Phenobarbital