This proposal contains five projects representing continuation of ongoing studies in the laboratory. The first project represents a continuation of our efforts to isolate, purify, and characterize the remaining forms of cytochrome P-450 of liver microsomes of the untreated rat. We have, to date, isolated eight forms. The methods used involve detergent solubilization of the microsomal membrane, separation of proteins on a jydrophobic column followed by cationic column chromatography, hydroxlapatite column chromatography and, orn occassion, anionic column chromatography. The purified proteins are characterized by NH2-terminal partialamino acid sequence, 2D-isoelectric focusingSDS-PAGE electrophoretograms, as well as by metabolite patterns with endogenous substrates like steriod hormones. In Project 2 the role of homeostasis on manitenance of constitutive forms of cytochrome P-450 is probed. Pathophysiological conditions are examined, e. g., diabetes, to determine effects on cytochrome P-450 rise and three forms decline. Insulin reverses these effects. The role of acetone as a mediator and its involvement in guconeogensis, under catalysis of RLM6, a diabetes induced P-450, will be examined. Cell culture conditions will be used to assess specific homeostatic regulators, e. g. hormones and chemicals. Protein-protein interactions will be studied in Project 3 and 4. In Project 3 individual protein modifiers and crosslinkers will be utilized to discern the mode of interaction of cytchrome P-450 with its redox partners. In Project 4, studies will be concerned with the mechanism of action of the P-450 system, concentrating on formation of electron transfer complexs of the redox proteins to study the electron flow patterns. The 5th project is a shorter study to assess the role of protein phosphorylation as a modulator of cytochrome P-450 in vivo. Conditions known to activate protein phosphorylation/dephosphorylation will be examined with respect to phosphorylation of individual forms of cytochrome P-450 and catalytic monooxygenase activity in hepatocyte cell preparation.
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