The experimental approaches described in this research proposal have been selected to elucidate the function(s) of the cytochrome P-450 omega- hydroxylase (P-450 PG-omega) in pulmonary microsomes which is highly induced by pregnancy, progesterone and other hormonal manipulations. This unique heme protein exhibits a substrate specificity toward eicosanoids with oxygen-containing functional groups at the omega-6 position and regioselectively hydroxylates these compounds at the omega- (or terminal) carbon. The gestational age dependence of this enzyme in the rabbit has prompted studies ranging from whole lung perfusion to determine metabolite profiles to isolation, purification, and characterization of the P-450 PG- omega at the molecular level. The hypothesis being tested is that P-450 PG-omega plays a protective role and/or participates in the triggering of parturition since ongoing studies in the Applicant's laboratory show definitive differences in whole lung metabolism of prostaglandins between the pregnant and non-pregnant animals. The molecular probes which the Principal Investigators's laboratory now has in hand will permit the further search for functional significance, elucidation of organ specificity, cellular localization, enzymatic mechanism, and metabolic control of these reactions. To this end, the following studies have been designed: 1) Localization of the cytochromes P-450 which catalyze these reactions in various organs and cells by immunohistochemical and immunoelectron microscopic techniques and in situ hybridization; 2) Measurement of the metabolism of the various eicosanoids (prostaglandins, hydroxyeicosatetraenoic acids, and thromboxanes) by regions and cell types of liver, lung, kidney, and placenta; 3) Determinations of substrate specificity, inhibition by specially designed mechanism-based inhibitors, and isolation of covalently bound tissue or purified enzyme adducts; 4) Assessment of physiological function of P-450 PG-omega or its orthologs in hemodynamic and cardiovascular measurements; and 5) full-length cDNAs in hand for screening of genomic clones, sequencing of cDNA and genomic clones, and expression in mammalian cells. The use of chimeric genes and site-directed mutagenesis will permit the determination of the sequence differences which contribute to the unique specificity of P-450 PG-omega and its orthologs, such as the fatty acid omega-hydroxylases.
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