[Oxidative modification of cytochrome P-450 during its function. I. Comparative study of the inactivation of cytochrome P-450 LM2 in various systems].

The changes in the content of purified isolated cytochrome P-450 LM2 under the action of hydrogen peroxide and during its operation in a soluble reconstituted system were studied. It was found that cytochrome P-450 LM2 inactivation by hydrogen peroxide is accompanied by a decrease in the hemoprotein activity, loss of heme, oxidation of SH-groups and changes in the oligomeric state of the enzyme. There were some differences in the mechanisms of cytochrome P-450 LM2 inactivation under the action of H2O2 and during catalysis.

Microheterogeneity of cytochrome P450(LM2) as revealed by isoelectric focusing.

The homogeneity of different preparations of cytochrome P450(LM2), the major form of cytochrome P450 found in the liver microsomes of phenobarbital-treated rabbits, was checked by SDS polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. In SDS-PAGE all the preparations migrated as a single protein band of molecular mass 49 kDa. The electrophoretic mobility of this band corresponded to that of the major phenobarbital-inducible band in the microsomes of phenobarbital-treated rabbits.

Effect of the microenvironment on the tertiary structure of cytochrome P-450 LM2.

The relation between microenvironment and the tertiary structure of cytochrome P-450 LM2 has been investigated. No complete relaxation to the most active state of the native enzyme took place in the case of membrane-incorporated hemoprotein with three or four intramolecular cross-links. The spatial organization of the enzyme was predicted to determine the cross-link location on the hemoprotein surface and membrane-incorporated parts of the polypeptide chain.

[The effect of microenvironment and formation of intra-molecular links on the tertiary structure of cytochrome P-450 LM2].

The effect of intramolecular cross-links formation in isolated cytochrome P-450 LM2 on its reactivation after incorporation into the liposome lipid bilayer was studied. Treatment with bifunctional reagents results in the inactivation of the solubilized haemoprotein. The degree of the enzyme immobilization determines the degree of inhibition of p-nitroanisol demethylation and aniline hydroxylation.

[Effects of hydrogen peroxide on cytochrome P-450 inactivation].

Inactivation rate of purified oligomeric cytochrome P-450 LM2 has been investigated in glucose oxidase system and under the action of exogenous hydrogen peroxide (400 microM). It has been found that hydrogen peroxide has a distinct inactivating effect on cytochrome P-450. The enzyme inactivation is accompanied by the loss of heme and the decrease in SH-group content in the protein molecule.