Purification and characterization of a new form (RLM2) of liver microsomal cytochrome P-450 from untreated rat.
A new cytochrome P-450 isozyme (RLM2) has been purified to electrophoretic homogeneity from liver microsomes of the untreated rat. It has an apparent minimum molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 49,000. Absolute spectrum of the oxidized form indicates that this isozyme is essentially all in the low spin state. The maximum of the reduced CO complex is at 449 nm. Amino-terminal partial amino acid sequence and amino acid composition are different from those of RLM3 and RLM5, two other native forms of cytochrome P-450 previously reported from this laboratory as well as other forms reported in the literature. RLM2 is capable of oxidizing a variety of drug substrates, like benzphetamine and aminopyrine, and to a lesser extent ethoxycoumarin. With the steroid substrate multiple isomeric products are formed differentially. Progesterone is preferentially hydroxylated at the 15-position (15 beta-hydroxylation (34%) and 15 alpha-hydroxylation (13%) of the total) and at the 6 beta-position (21%). The major metabolite when testosterone was the substrate, 15 alpha-hydroxytestosterone, comprised 43% of the total, while a modest amount of 6 beta-hydroxytestosterone (12%) is formed. Another major metabolite (31%) has yet to be unequivocally identified, but is suggested to be 7 beta-hydroxytestosterone. Examination of the substrate dependence of major and minor isomeric metabolites provides evidence for a single substrate-binding site on RLM2. Regardless of the position hydroxylated, a common Km value was obtained. It is suggested that differences in formation of the isomeric and epimeric products relate to differences in distance from the active oxygen center and the position of attack.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002852:Chromatography, Ion Exchange; D002855:Chromatography, Thin Layer; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D008401:Gas Chromatography-Mass Spectrometry; D007527:Isoenzymes / Q000302:isolation & purification; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D011374:Progesterone / Q000378:metabolism; D051381:Rats; D013739:Testosterone / Q000378:metabolism
D000596:Amino Acids; D007527:Isoenzymes; D013739:Testosterone; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System