When this research project was initiated in 1984, the experimental focus was on the purification and characterization of the prostaglandin w- hydroxylase induced more than 100-fold in the lungs of pregnant rabbits, first observed by Powell and his co-investigator [J. Biol. Chem., 253, 6711-6716(1978)]. This laboratory [Williams, et al., J. Biol, Chem., 259, 14600-14608 (1984)], coincident with that of Kusunose in Japan [Yamamoto, et al., J. Biochem, (Tokyo), 96, 593-603 (1984)] purified the enzyme catalyzing this activity and characterized it as cytochrome P450. This enzyme has been classified as a member of the cytochrome p450 gene subfamily, CYP 4A, and designed CYP 4A4, indicating its homology with the rat liver laureate w-hydroxylase cloned ad sequenced by Hardwick, et al. [J. Biol. Chem, 262, 801-810 (1987)]. There are now at least 12 members of this gene subfamily, several of which have been cloned, sequenced and expressed in collaboration with Dr. Eric Johnson's laboratory. The expression of these clones in African green monkey kidney cells (COS-1) has permitted the determination of relative substrate specificities but not the establishment of catalytic efficiencies or physical properties. A number of these CYP 4A proteins have been shown to catalyse the w- hydroxylation of arachidonic acid to form 20-hydroxyeicosatetraenoic acid (20-HETE), a potent vasoconstrictor of renal arcuate arteries and aortic rings.
The Specific Aims of this proposal are designed to address those properties of the various members of this cytochrome p450 gene subfamily, p450 4A4 (lung) and P450s 4A5, 4A6, and 4A7 (kidney), which confer the substrate specificities and determine structure-function relationships, and to identify those cytochromes P450 catalyzing the formation of arachidonic acid metabolites producing specific physiological effects.
Specific Aims are: 1) to express cytochromes P450 4A4, 4A5, 4A6, and 4A7 in E. coli in order to obtain sufficient quantities for kinetic, spectral, and biophysical characterization; 2) to utilize discriminatory inhibitory inhibitors, designed as mechanism-based, """"""""suicide substrates"""""""", which can applied in vivo, as well as in vitro, to probe the functions of these enzymes; 3) to determine, by site-directed mutagenesis, the specificity-conferring determinants/domains of cytochromes P450 4A4, 4A5, 4A6, and 4A7, utilizing the information obtained in our laboratory with chimeric constructs and any additional information obtained in structure- function studies of these and other cytochromes P450; 4) to perform in situ hybridization for differential determination of the localization of the cytochrome P450 4A proteins and to develop cell culture models which will mimic the inductive effects of hormonal treatments of whole animals, e.g., progesterone and dexamethasone, to study the regulation of the lung and kidney CYP 4A gene subfamily members, which are differentially induced; and 5) to develop transgenic animal models to determine the function(s) of the lung (P450 4A4) and kidney (P450s 4A5, 4A6, 4A7) enzymes, for example in maintaining and regulating hemodynamic homeostasis. These studies are designed to address those properties of the members of this closely related gene subfamily which determine their substrate specificities and, thus, to ascertain their functional significance. It is particularly noteworthy that the various CYP 4A proteins are differentially regulated by hormonal treatment of whole animals. The regulation of hemodynamics in the kidney by arachidonic acid metabolites is a subject of current interest and intense research regarding the pathophysiology of acquired and genetically determined hypertension.
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