The long range goal of the proposed research is to determine the disposition in the membrane and the mode of interaction of cytochrome P-450 and NADPH-cytochrome P-450 reductase of the microsomal mixed function oxidase system. These enzymes participate in the inactivation of many natural metabolites, drugs and other xenobiotics, and in tumorigenesis and liver degeneration induced by several organic environmental pollutants and industrial chemicals.
The specific aims are: (1) determination of the mechanism of interaction in microsomes, lipid-suplemented microsomes and in liposomes between cytochrome P-450 and NADPH-cytochrome P-450 reductase--i.e., do these enzymes interact with each other during catalysis through random encounters as they diffuse laterally in the membrane or do they form a stable intermolecular complex? (2) determination of the amino acid residues of cytochrome P-450 and NADPH-cytochrome P-450 reductase which interact during catalysis; (3) determination of the cytochrome P-450 isozymes exist as aggregates or monomers in microsomes, and if aggregates are found, identification of the isozymes involved in aggregation. Experimental approaches to be used include reconstitution of purified cytochrome P-450 isozymes into active systems containing either non-vesicular phospholipid, phospholipid vesicles or detergents. Chemical cross-linking agents will be used to assess aggregation in solution, in artifical vesicles, in lipid-supplemented microsomes, and in microsomes. Immunochemical techniques with monospecific antibodies will be used to identify the cross-linked species after separation of the components by polyacrylamide gel electrophoresis under denaturing conditions. Comparison of the profiles of cross-linked species obtained with these systems will allow definition of the appropriate model of P-450/reductase interaction: complex formation or free diffusion. Identification of the site of interaction between reductase and P-450 will make use of a novel site-directed photoactivatable affinity label consisting of an appropriate derivative of the reductase which is known to specifically interact with cytochrome P-450. Understanding the mechanism of interaction between these two proteins will enhance our understanding not only of xenobiotic metabolism, but also of the interaction between membrane-associated proteins in general.
| Wagner, S L; Dean, W L; Gray, R D (1987) Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase. Biochemistry 26:2343-8 |
| Gray, R D; Dean, W L (1987) The effect of 2,3-diphosphoglycerate on the tetramer-dimer equilibrium of carbon monoxide hemoglobin in dilute solution. Correlation between sedimentation and kinetic behavior. J Biol Chem 262:15890-3 |
