Kinetics of rat liver microsomal estrogen 2-hydroxylase. Evidence for sex differences at initial velocity conditions.
The enzyme kinetics of estrogen 2-hydroxylase from rat liver microsomes has been investigated using two radiotracer methods. The conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol by microsomes was examined by isolation of the catechol estrogen. [6,7-3H]2-Hydroxyestradiol was prepared and added at the end of the incubation as a recovery marker for quantitation. Initial velocity conditions of less than 10% product formation were established. The enzyme kinetics for estrogen 2-hydroxylase from male rat liver follows classical Michaelis-Menten kinetics producing a linear Lineweaver-Burk plot. The apparent Km for estradiol under these conditions was 2.2 microM; the Vmax for freshly prepared microsomes was 5.0 nmol/mg/min while that for microsomes stored at -70 degrees C was 0.51 nmol/mg/min. On the other hand, estrogen 2-hydroxylase from female rat liver microsomes did not follow Michaelis-Menten kinetics and yielded a nonlinear double reciprocal plot. Analogous kinetic data were obtained with another radiotracer assay measuring the release of 3H2O from [2-3H]estradiol. Thus, the estrogen 2-hydroxylases of male and female rat liver differ in enzyme kinetic properties. The upwardly curving Lineweaver-Burk plot of the kinetic data of the female rat liver microsomes suggests that more than one binding site for estradiol may exist on this enzyme complex and/or that this particular estrogen 2-hydroxylase system exhibits cooperative effects.
D000818:Animals; D002250:Carbon Radioisotopes; D019363:Cytochrome P-450 CYP1A1; D004967:Estrogens / Q000378:metabolism; D005260:Female; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012737:Sex Factors; D013250:Steroid Hydroxylases / Q000378:metabolism*
D002250:Carbon Radioisotopes; D004967:Estrogens; D013250:Steroid Hydroxylases; D019363:Cytochrome P-450 CYP1A1; C020120:estrogen 2-hydroxylase