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Biomedical subjects

A V Karyakin

Publications and source records attributed to A V Karyakin.

8 recordsLinked to original sources
PubMed1986

Reconstitution of liver monooxygenase system in solution from cytochrome P-450 and NADPH-specific flavoprotein monomers.

Microsomal monooxygenase system was reconstituted in the presence of non-ionic detergent Emulgen 913 from cytochrome P-450 and NADPH-specific flavoprotein isolated from phenobarbital-induced rabbit liver microsomes. At Emulgen 913 concentration of 0.05 g/l mixed complex between flavoprotein and cytochrome was formed with 5: 5 protein molar ratio and molecular weight of 700 kD. The 2-hour incubation of the enzymes with 0.25 g/l Emulgen 913 at 4 degrees C was accompanied by dissociation of protein oligomers to monomers. The reconstituted systems containing flavoprotein and cytochrome as mixed complexes or monomers were able to catalyze NADPH-dependent cytochrome P-450 reduction and benzphetamine N-demethylation. Taking into consideration the effective concentrations of the enzymes the apparent second order rate constants of these reactions with monomers were 100 times those with complexes.

Animals↗
PubMed1985

Kinetic studies on reduction of cytochromes P-450 and b5 by dithionite.

The kinetics of reduction of cytochromes P-450 and b5 by dithionite had been studied in solution and in microsomal and proteoliposomal membranes by the stopped-flow technique. In all the cases studied the kinetic curves of reduction of cytochrome b5 obey first-order kinetics in relation to cytochrome with the rate constant about 14 s-1 at 10.6 mM dithionite. The kinetic curves of reduction of cytochrome P-450 fit an equation for the sum of two exponentials with the parameters varying from system to system. The simple first-order kinetics of cytochrome P-450 reduction had been observed in the presence of non-ionic detergent Triton N-101. The apparent biphasity of cytochrome P-450 reduction by dithionite should be the result asymmetric distribution of hemoprotein in microsomal and proteoliposomal membranes as well as in cytochrome oligomers in solution.

Animals↗
PubMed1983

The influence of cholesterol incorporation and removal on lipid-bilayer viscosity and electron transfer in rat-liver microsomes.

The incorporation and removal of cholesterol from rat liver microsomes was used as a methodical approach to investigate the molecular organization of microsomal redox-chains. It was shown that the incorporation of cholesterol in microsomes increases and removal of cholesterol decreases lipid bilayer viscosity as indicated from the rate of fluorescent probe-pyrene eximerisation in cholesterol-enriched and cholesterol-depleted microsomes. The increase of membrane viscosity slows down the initial rates and decreases the rate constants of cytochrome b5 reduction by NAD(P)H, whereas the decrease of membrane viscosity enhances the initial rates and increases the rate constants of these reactions. The rates of cytochrome P450 reduction by reduced pyridine nucleotides do not depend on the viscosity of lipid bilayer. The incorporation and removal of cholesterol from microsomes was not followed by any essential changes in the rates of dimethylaniline N-demethylation, aniline p-hydroxylation, p-nitroanisole O-demethylation, oxygen consumption, oxidation of NADH and NADPH. Thus the reduction of cytochrome b5 by NADH and NADPH is the diffusion-dependent reaction in the redox-chains of microsomes only.

Animals↗
PubMed1975

Intermembrane electron transport in the absence of added water-soluble carriers.

Electron transport from untreated to mersalyzed microsomal vesicles at the level of NADH-cytochrome b5 reductase or cytochrome b5 has been demonstrated in the absence of added water-soluble electron carriers. A similar effect was shown in the systems "intact mitochondria - mersalyzed microsomes" and "mersalyzed mtiochondria - untreated microsomes". No measurable electron transport between intact and mersalyzed particles of inner mitochondrial membrane was found. The obtained data suggest that the capability to carry out intermembrane electron transfer is specific for NADH-cytochrome b5 reductase and/or cytochrome b5, localized in microsomal and outer mitochondrial membranes.

Aerobiosis↗
PubMed1974

The reconstitution of microsomal redox chains. A comparitive analysis of the effectiveness of membrane self-assembly and template binding of electron carriers.

A comparative analysis was made of the effectiveness of three methods for the reconstitution of microsomal electron-transfer chains, namely, self-assembly, incorporation of electron carriers into liposomes (non-specific template) and incorporation into ;ghosts' of microsomal vesicles (specific template). It was shown that when the ;ghosts' of the microsomal vesicles were used as a specific template extra cytochrome b(5) and NADH-specific flavoprotein were incorporated into them, but cytochrome P-450 and NADPH-specific flavoprotein were not incorporated into the membrane. As a result of the self-assembly and incorporation into liposomes all the electron carriers were present in the reconstituted membrane. Cytochrome P-450 reactivation took place and the inactive form, cytochrome P-420, was converted into the active form, cytochrome P-450. Of the four enzyme hydroxylation systems studied, i.e. NADPH- and NADH-dependent p-hydroxylation of aniline, and NADPH- and NADH-dependent N-demethylation of dimethylaniline, only the NADH-dependent demethylation of dimethylaniline (60% of the initial value) and NADH-dependent p-hydroxylation of aniline (30% of the initial value) were reconstituted by self-assembly. NADPH oxidase and NADH oxidase activities were only properly reconstituted by self-assembly and incorporation into liposomes. In contrast, the NADPH-specific system of peroxidation of unsaturated fatty acids was reconstituted by specific template-binding.

Animals↗
PubMedDate not supplied

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes.

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.

Animals↗