Flavin-containing monooxygenase (FMO, EC1.14.13.8) comprises a family of xenobiotic-metabolizing enzymes that catalyze the oxygenation of a wide variety of xenobiotics, most commonly nitrogen and sulfur. Mammals express five different FMOs in a species- and tissue- specific manner. FMO2, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxygenation of certain primary alkylamines. From its initial discovery as an unique form of FMO in lung, FMO2 has been studied primarily using a rabbit animal model. The initial goal of this research was to characterize this protein in a primate animal model. To understand FMO2 protein structure at the molecular level, we first cloned cDNA from a monkey lung cDNA library. Monkey FMO2 is expressed predominantly in lung. The high expression levels and broad substrate specificity in monkey, suggests that FMO2 plays a role in xenobiotic metabolism in this primate model. We then established a heterologous expression system to generate FMO2 with biological functionality in vitro. FMO2 from baculovirus-mediated expression resembled monkey and rabbit microsomal FMO2 immunochemically and catalytically. The successful FMO2 expression in the baculovirus system will serve as a valid tool for structure studies of protein functional domains, as well as, the amino acids responsible for enzyme properties of chimeras. Human FMO2 encodes a truncated protein containing 471 amino acid residues, 64 amino acids shorter in its C-terminal than orthologs in other species. We characterized human FMO2 in terms of gene polymorphism (genotyped by Dr. Hines), protein levels and catalytic activity with human lung microsomes. An ethnically related polymorphism was observed, in which all Caucasians genotyped to date are homozygous for a truncated, enzymatically inactive enzyme which may not even be translated. Approximately 15% of humans of African descent are heterozygous for full-length FMO2, but the level of expression may not be sufficient to significantly effect drug metabolism in the lung. The results of truncated FMO2 produced from baculovirus expression suggest that the C-terminal of FMO2 might be responsible for enzyme stability and/or proper structure necessary to exert fully enzyme activity. We conclude that the human FMO2 possesses unique features compared to all other mammals examined to date including other primates. / Graduation date: 1999

This study was designed to evaluate the possible existence of masked gonadotropin binding sites in the corpus luteum of the rhesus monkey. Pretreatment of macaque luteal particulates and cells with neuraminidase increased LH binding. In vitro exposure to alcohols also enhanced LH binding to these preparations. Ethanol modulation of LH binding was a time- and temperature-dependent process. The optimal concentration of ethanol for enhancing LH uptake was inversely proportional to the incubation temperature. Longer straight-chain alcohols were more potent than ethanol in increasing LH binding. Ethanol and neuraminidase increased the number of binding sites with no affect on affinity. Moreover, the effects of ethanol and NA were additive. Since alcohols and temperature are modulators of membrane fluidity, we examined the hypothesis that the unmasking of gonadotropin binding sites may be related to changes in the fluid state of the lipid bilayer of the luteal membrane. First, membrane fluidity was estimated from the fluorescence polarization of the membrane probe diphenylhexatriene. Conditions which resulted in enhanced gonadotropin binding (1-8% ethanol, increased temperature), increased the fluidity of luteal membranes. Moreover, changes in gonadotropin binding were highly correlated (r = -0.97) with changes in membrane fluidity under these conditions. Pretreatment of luteal particulates with neuraminidase had no apparent effect on membrane fluidity. Second, gonadotropin receptors were removed from the luteal membrane by detergent solubilization, and the effects of ethanol on soluble receptors were compared to those on receptors associated with the lipid bilayer. Solubilization resulted in the recovery of 50% more gonadotropin binding sites than are available in particulate preparations of the corpus luteum; these sites displayed lower affinity for gonadotropin. Moreover, conditions which increase LH binding to luteal particulates (1-8% ethanol at 25C) decreased LH uptake by soluble receptors. The data suggest that two populations of LH binding sites are masked within the membranes of the monkey corpus luteum. The ability of two markedly different agents, alcohol and neuraminidase, to increase LH binding indicates the diverse mechanisms may modulate the masking/unmasking of gonadotropin receptors in target cell membranes. As such, changes in membrane fluidity may play an important role in this response.

After vasectomy, the passage of sperm antigen through the epithelium of the efferent duct in the rhesus macaque probably results in immune complex deposition. Immune complexes can be visualized in the thickened basement by fluorescence microscopy. Subsequent electron microscopic evaluation, with the appearance of electron-dense deposits in the basement membrane of the efferent ducts, substantiates these findings.