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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM026114-12
Application #
3273607
Study Section
Pharmacology A Study Section (PHRA)
Project Start
1978-06-01
Project End
1993-05-31
Budget Start
1989-06-01
Budget End
1990-05-31
Support Year
12
Fiscal Year
1989
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Type
School of Medicine & Dentistry
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Jansson, I; Mole, J E; Schenkman, J B (1995) The isolation and comparison of multiple forms of CYP2B from untreated and phenobarbital-treated rabbit liver microsomes. Arch Biochem Biophys 316:275-84
Voznesensky, A I; Schenkman, J B; Pernecky, S J et al. (1994) The NH2-terminal region of rabbit CYP2E1 is not essential for interaction with NADPH-cytochrome P450 reductase. Biochem Biophys Res Commun 203:156-61
Voznesensky, A I; Schenkman, J B (1994) Quantitative analyses of electrostatic interactions between NADPH-cytochrome P450 reductase and cytochrome P450 enzymes. J Biol Chem 269:15724-31
Schenkman, J B; Voznesensky, A I; Jansson, I (1994) Influence of ionic strength on the P450 monooxygenase reaction and role of cytochrome b5 in the process. Arch Biochem Biophys 314:234-41
Richardson, T H; Schenkman, J B; Turcan, R et al. (1992) Molecular cloning of a cDNA for rat diabetes-inducible cytochrome P450RLM6: hormonal regulation and similarity to the cytochrome P4502E1 gene. Xenobiotica 22:621-31
Voznesensky, A I; Schenkman, J B (1992) The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing. J Biol Chem 267:14669-76
Voznesensky, A I; Schenkman, J B (1992) Inhibition of cytochrome-P450 reductase by polyols has an electrostatic nature. Eur J Biochem 210:741-6
Cammer, W; Downing, M; Clarke, W et al. (1991) Immunocytochemical staining of the RLM6 form of cytochrome P-450 in oligodendrocytes and myelin of rat brain. J Histochem Cytochem 39:1089-94
Schenkman, J B (1991) Induction of diabetes and evaluation of diabetic state on P450 expression. Methods Enzymol 206:325-31
Sinclair, J F; McCaffrey, J; Sinclair, P R et al. (1991) Ethanol increases cytochromes P450IIE, IIB1/2, and IIIA in cultured rat hepatocytes. Arch Biochem Biophys 284:360-5

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