Purification, characterization, and pituitary regulation of the sex-specific cytochrome P-450 15 beta-hydroxylase from liver microsomes of untreated female rats.
A sex-differentiated form of cytochrome P-450 has been purified to electrophoretic homogeneity from liver microsomes of untreated female rats. The purified preparation contained 17 nmol of P-450/mg of protein and had a minimum molecular weight of 50,000. The final preparation was active in the hydroxylation of 5 alpha-[3H]androstane-3 alpha, 17 beta-diol 3,17-disulfate in the 15 beta position, with a turnover number of 2.6 nmol/min/nmol P-450 and is designated "P-450 15 beta" on the basis of this activity. Cytochrome P-450 15 beta is isolated essentially in the low-spin state and has a CO-reduced difference spectral maximum at 449 nm. Comparison of the female protein with the corresponding P-450 fraction from male rats revealed an absence of the 15 beta band in the male electrophoretic profile. Specific antibodies to isozyme 15 beta were used with a Western blot technique to demonstrate the virtual absence of the protein in male microsomes. This method was also used to demonstrate that hypophysectomy of female rats resulted in undetectable levels of the 15 beta-hydroxylase, while continuous infusion of growth hormone to normal male animals increased the 15 beta-hydroxylase level to that of female. P-450 15 beta is proposed to be the enzyme responsible for the predominance of 15 beta-hydroxylated steroid sulfate metabolites in excreta of female rats, and their absence in males. The same purification procedure for female rat liver microsomes also yielded another purified cytochrome P-450 characterized by a minimum molecular weight of 52,500, termed P-450 DEa, which was inefficient in the 15 beta-hydroxylation of 5 alpha-[3H]androstane-3 alpha, 17 beta-diol 3, 17-disulfate. No evidence was obtained that this form is sexually differentiated.
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D005260:Female; D007016:Hypophysectomy; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010902:Pituitary Gland / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D013053:Spectrophotometry; 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; C034694:steroid 15-beta-hydroxylase