Isolation and partial characterization of a cytochrome P-450 isoenzyme (cytochrome P-450tu) from mouse liver tumors.
Experimental hepatomas induced with 5,9-dimethyldibenzo[c,g]carbazole in female XVIInc/Z mice display a strong microsomal steroid 15 alpha-hydroxylation activity. A cytochrome P-450 isoenzyme (cytochrome P-450tu), specific for this activity, has been isolated by an HPLC derived method using various Fractogel TSK and hydroxyapatite supports. On SDS polyacrylamide gel electrophoresis the purified protein appeared as one major band with an apparent Mr of 50,000. Its specific cytochrome P-450 content was 7.55 nmol/mg protein. As deduced from the visible spectrum, the heme iron of the isolated P-450tu was to 72% in the high-spin state. The CO-bound reduced form showed an absorption maximum at 450 nm. In addition to the stereospecific 15 alpha-hydroxylation of progesterone (2.3 min-1) and testosterone (2.5 min-1), the enzyme catalyzed also 7-ethoxycoumarin O-deethylation, benzphetamine N-demethylation and aniline 4-hydroxylation. Its N-terminal amino-acid sequence (21 residues) was identical to that of cytochrome P-450(15) alpha, isolated by Harada and Negishi from liver microsomes of 129/J mice. P-450tu differed from P-450(15) alpha by its higher molecular weight, its 40-times lower steroid 15 alpha-hydroxylation and its 4-times higher benzphetamine N-demethylation.
Lange
R
R
Institut National de la Santé et de la Recherche Médicale U 128, Montpellier, France.
Périn
F
F
Larroque
C
C
Zajdela
F
F
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002852:Chromatography, Ion Exchange; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification* / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D013057:Spectrum Analysis; D013250:Steroid Hydroxylases / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity
D007527:Isoenzymes; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C050356:steroid 15-alpha-hydroxylase