RABBIT LUNG FLAVIN-CONTAINING MONOOXYGENASE - PURIFICATION, CHARACTERIZATION, AND INDUCTION DURING PREGNANCY

Abstract

A flavin-containing monooxygenase has been purified to apparent homogeneity from lung microsomes of pregnant rabbits and characterized with respect to a number of physical and catalytic parameters. The apparent molecular weight, as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 59,000, and the lung microsomal flavoprotein was shown to contain 14 nmol of FAD/mg of protein. Addition of NADP+ to the oxidized flavoprotein produced a shift in the spectrum characteristic of the flavin-containing monooxygenase from porcine liver, and addition of small amounts of NADPH to the oxidized rabbit lung enzyme produced a stable spectral intermediate consistent with that of a 4a-peroxyflavin. Rabbit lung flavin-containing monooxygenase differed markedly from the porcine liver enzyme in exhibiting a broader pH optimum from 8.5-10.5, by not being inhibited by concentrations of sodium cholate as high as 1% and by withstanding, in the absence of NADPH, incubation at 45.degree. for at least 10 min with no significant loss of activity. Unlike the pig liver enzyme, purified rabbit lung enzymes was not activated by n-octylamine and, in fact, n-octylamine stimulated NADPH oxidation. A number of compounds known to be substrates of the pig liver enzyme, including benzphetamine, chlorpromazine, and imipramine, are not substrates for the rabbit lung enzyme, whereas prochlorperazine and trifluoperazine are excellent substrates. Antibodies to rabbit lung flavin-containing monooxygenase were raised in guinea pig and utilized for the immunoquantitation of this enzyme throughout gestation. The pig and utilized for the immunoquantitation of this enzyme throughout gestation. The activity (as determined by N,N-dimethylaniline-N-oxidation) and amount of rabbit flavin-containing monooxygenase were maximally induced (5-fold) on the 28th day of gestation. Liver microsomes from rabbit did not contain any of the lung form of flavin-containing monooxygenase at any time during gestation, as evidenced by results from Western blotting. These results demonstrate that, at least in rabbit, flavin-containing monooxygenase can exist as more than a single form. The physiological significance of the induction of this enzyme during pregnancy in not known.