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Biomedical subjects

A I Archakov

Publications and source records attributed to A I Archakov.

At least 19 recordsLinked to original sources
PubMed1992

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. I. Properties of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) monomers.

Oligomers and monomers of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 (2B4) isolated from the liver microsomes of phenobarbital-treated rabbits were examined for physicochemical properties and catalytic activities. As measured using laser correlation spectroscopy the particle sizes of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers were 14.8 +/- 1.7 and 19.2 +/- 1.4 nm, respectively. Twenty-four-hour incubation with Emulgen 913 at 4 degrees C at a molar ratio of 1:100 led to the monomerization of NADPH-cytochrome P450 reductase and cytochrome P450 LM2 oligomers, the particle sizes diminishing to 6.1 +/- 1.3 and 5.2 +/- 0.4 nm, respectively. The thermal stability of NADPH-cytochrome P450 reductase monomers was the same as that of oligomers, whereas cytochrome P450 LM2 monomers were less thermostable than oligomers and cytochrome P450 in microsomes. Similar to cytochrome P450 LM2 oligomers and the microsomal hemoprotein, cytochrome P450 LM2 monomers formed complexes with type I and II substrates, but with Kd values higher than those of microsomes and cytochrome P450 LM2 oligomers. Kinetic parameters (Vmax and Km) of H2O2- and cumene hydroperoxide-dependent oxidation of benzphetamine and aniline in the presence of cytochrome P450 LM2 oligomers, monomers, and microsomes were determined. Peroxidase activities of the oligomers and monomers were the same, but were lower than those of microsomes. Thus the substitution of protein-protein interactions in cytochrome P450 LM2 oligomers with protein-detergent interactions in the monomers did not influence the catalytic properties of the hemoprotein.

Aniline Compounds↗
PubMed1992

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.

Animals↗
PubMed1992

Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers.

The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.

Amino Acid Sequence↗
PubMed1991

Effects of cholesterol- or 7-ketocholesterol-containing liposomes on colony-forming ability of cultured cells.

Experiments with cultured Chinese hamster cells showed that incubation of the cells with (phosphatidylcholine + cholesterol + 7-ketocholesterol)-containing liposomes (4:3:1 by weight) during two hours led to a decrease in the colony-forming ability of cells down to zero, while (phosphatidylcholine + cholesterol)-containing liposomes (1:1 by weight) reduce this parameter by 90%. Furthermore, the cholesterol-containing liposomes (without 7-ketocholesterol) induce a decrease in the number of the maximal-site colonies accompanied by the corresponding increase in the number of the middle-size colonies.

Animals↗
PubMed1991

Cytochrome P-450 spin state and leakiness of the monooxygenase pathway.

1. The monooxygenase and oxidase activities of liver microsomes from phenobarbital (PB)-treated rabbits were investigated for their dependence on the high spin shift (delta alpha) of the ferric cytochrome P-450 induced by a series of benzphetamine analogues. 2. The spin shift activity of the substrate determines, via the first electron transfer kinetics, the steady-state level of the reaction intermediate oxycytochrome P-450. Correlation of the amount or oxycytochrome P-450 with delta alpha can be experimentally proved. 3. The spin-state-dependent formation of oxycytochrome P-450 regulates quantitatively the rates of NADPH oxidation and substrate N-demethylation. Both activities correlate with delta alpha. Oxycytochrome P-450 is substrate-stabilized towards decay with the formation of O2- which, upon dismutation, gives rise to H2O2. 4. The ratio of N-demethylase to NADPH oxidase activity (coupling ratio) also increases with the spin shift, delta alpha. Concomitantly, the proportion of NADPH accounted for by H2O2 and H2O formation via two- and four-electron reduction of dioxygen decreases. This indicates that the substrate-induced structural changes in the enzyme active centre which give rise to spin transition may likewise modify the coupling properties. 5. Perfluorinated compounds, which fail to undergo monooxygenation, fall in line with the benzphetamine derivatives with respect to the dependence of NADPH oxidation rate and steady-state oxycytochrome P-450 level on delta alpha. The increased oxidase activity results mostly in H2O formation. 6. The leakiness of the PB-induced monooxygenase pathway in the biotransformation of oxygen in the presence of the benzphetamines and perfluorinated compounds does not result in marked increases in H2O2 formation. Therefore, the increase of NADPH oxidase activity by these substrates does not significantly enhance H2O2-mediated oxygen tissue toxicity.

Animals↗
PubMed1991

Covalent binding of bleomycin to concanavalin A and immunoglobulin G enhances the ability of the bleomycin-Fe(II) complex to destroy the erythrocyte membrane.

The antibiotic bleomycin was examined as a possible component of hybrid molecules composed of an address fragment and a generator of reactive oxygen species. The bleomycin-Fe(II) complex was found to destroy the erythrocyte membrane by generating reactive oxygen. The ability of antioxidants to slow down haemolysis points to a free-radical mechanism for this process. The protective effects of catalase and superoxide dismutase indicate that hydrogen peroxide and the superoxide radical formed on autoxidation of the complex are essential for membrane damage. Haemolytic activity is also exhibited by bleomycin-Fe(III) reduced in the NADPH-cytochrome P450 reductase reaction. The covalent binding of bleomycin to such address molecules as concanavalin A and antierythrocyte immunoglobulin G enhances the ability of the bleomycin-Fe(II) complex to destroy the plasma membrane of erythrocytes.

Animals↗
PubMed1991

Diet-induced hypercholesterolaemia in the rabbit.

Intraspecies variation in diet-induced hypercholesterolaemia in rabbits, simulating atherosclerosis, was studied. The chinchilla rabbit population examined contained several subpopulations, as indicated by polymodal forms of histograms of plasma cholesterol levels. This finding indicates that the inclusion of subpopulations in an investigation can lead to erroneous conclusions, and that subpopulations should be identified before such work is undertaken. The relationship between the molar cholesterol/phospholipid ratio in rabbit erythrocyte membranes and the plasma cholesterol level in experimental atherosclerosis was also studied. A correlation was evident only over the range of 1-5 g cholesterol per litre of plasma.

Animals↗
PubMed1991

Interactions of cholesterol-containing liposomes with serum lipoproteins.

The interaction of liposomes containing different amounts of cholesterol with low-density lipoproteins from human serum was investigated. The efficiency of the interaction was found to depend on the cholesterol content of the liposomes and was highest for liposomes with the maximum cholesterol:phospholipid molar ratio. These liposomes selectively and effectively interacted with low-density lipoproteins; up to 90% of lipoprotein particles interacted with liposomes in serum. Fusion of particles with liposomes was observed during the interaction.

Cholesterol↗
PubMed1991

[Induction of cytochrome P-450 by triterpensaponins in Vietnamese ginseng].

It was found that rat liver cytochrome P-450 is induced by the Vietnamese ginseng triterpensaponines mixture (TSM) as well as by K5VN Panaxozides-11 (VP-11) purified from this mixture. Addition of TSM and VP-11 accelerates benz(alpha)pyrene and aminopyrine hydroxylation and increases the content of cytochrome P-450 isoforms with Mr of 57 kDa and 54 kDa in rat liver microsomes. Since VP-11 accounts for about 50% of TSM, the results obtained suggest that the microsomal monooxygenase system induction is caused by this triterpensaponine. Induction by TSM and VP-11 was compared to that by phenobarbital (PB) and 3-methylcholanthrene (MC). It was shown that according to their inductive action TSM and VP-11 belong neither to the PB- nor to the MC-type. Cytochrome P-450 induction may play an important role in the triterpensaponine action on the organism, because this enzyme participates in the metabolism of such endogenous compounds as prostaglandins, steroid hormones, cholesterol, etc.

Aminopyrine↗
PubMed1991

[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.

Animals↗
PubMed1991

[Oxidative modification of cytochrome P-450 during its function. II. Study of the mechanism of cytochrome P-450 LM2 inactivation in a soluble reconstructed monooxygenase system].

Inactivation of cytochrome P-450 LM2 induced by hydrogen peroxide formed in the active site of the enzyme was studied. Catalase did not protect cytochrome P-450 LM2 from inactivation during its operation in a soluble reconstituted system. The hemoprotein inactivation in this system was found to depend on the ratio of hemo- to flavoproteins. It was demonstrated that cytochrome P-450 LM2 inactivation during catalysis is accompanied by cleavage of the hemoprotein molecule. It is probable that this fact plays a key role in regulation of enzyme decay.

Animals↗
PubMed1990

Conjugation of bleomycin with concanavalin A or immunoglobulin G increases its ability to destroy cell membranes.

Hybrid molecules consisting of an address peptide and an active oxygen-generating fragment may be used for selective destruction of cells. We tested the possibility of using the antibiotic bleomycin (BLM) as an active oxygen-generating fragment of such a molecule. It was found that bleomycin can induce destruction of cell membranes. BLM-mediated cell destruction was inhibited by addition of catalase, superoxide dismutase, and OH. scavangers (mannitol and ethanol), suggesting that hydroxy radical is involved in the process. BLM can induce membrane impairment at the expense of electrons supplied by NADPH-cytochrome P450 reductase. Covalent binding of BLM to an address fragment (concanavalin A, immunoglobulin G) increases the ability of BLM to destroy erythrocyte membranes. The data obtained lead to the conclusion that BLM can be used as an active oxygen-generating fragment of a proposed cell-destroying hybrid molecule.

Animals↗
PubMed1979

[Comparative study of effects of the tyrosine-copper(II) complex on xenobiotic hydroxylation and lipid peroxidation].

It has been found that NADPH-dependent hydroxylation of dimethylaniline, aniline, p- and o-nitroanisol and lipid peroxidation is inhibited by the tyrosine-copper (II) complex (low molecular weight analog of superoxide dismutase), which is indicative of a possibility of superoxide radicals formation in these reactions. The inhibition of the above-mentioned reactions with Tyr2-Cu2+ is less pronounced or absent, if cumole hydroperoxide is used as cosubstrate instead of NADPH. Differences in the Tyr2-Cu2+ complex effects on the cumule hydroperoxide-dependent xenobiotics hydroxylation and lipid peroxidation catalyzed by various forms of cytochrome P-450, e. g. microsomal, soluble and incorporated into liposomes, have been found. The data obtained suggest that the efficiency of the inhibitory effect of the Tyr2-Cu2+ complex depends on the type of cosubstrates (NADPH, cumole hydroperoxide) and substrates used as well as on the form of cytochrome P-450.

Animals↗
PubMed1979

[Accessibility of microsomal membrane proteins for protease K].

The effect of protease K on the microsomal membrane proteins was studied. It was shown that treatment of the microsomal membranes by low concentrations of protease did not remove more than 35% of total membrane proteins. Treatment by higher protease concentrations removed about 50% of the proteins. The residual protein is accessible to protease after treatment of the membranes by phospholipase A2. A simultaneous treatment of the microsomal membrane by protease and phospholipase removed up to 80% of the membrane proteins. In this way about 30% of microsomal membrane proteins are protected by the phospholipid against the action of protease K.

Animals↗
PubMed1979

[Microsomal hemoprotein reduction by superoxide radical formed on NADPH-specific flavoprotein].

The superoxide radicals formed on NADPH-specific flavoprotein of liver microsomes can reduce cytochromes c, b5, and P-450. This reaction is inhibited under aerobic conditions by a low molecular weight analog of superoxide dismutase, e.g. the copper-tyrosine complex. The inhibitory effect of the complex is not observed under anaerobic conditions. Based on the results obtained a scheme of the electron transfer between the flavoprotein and haemoproteins involving superoxide radicals is proposed.

Aerobiosis↗
PubMed1979

[Reconstitution of liver endoplasmic reticulum membranes from solubilized proteins and lipids by removal of detergent].

A method for membrane reconstitution from cholate-solubilized microsomal proteins and lipids by a removal of the detergent on a column with charcoal has been developed. A comparative study showed that the membranes reconstituted by a dialysis or absorption do not differ from each other in terms of membrane proteins incorporation into lipid vesicles and cytochrome P-450 reconversion into cytochrome P-450. A possibility of biomembrane reconstitution from membrane proteins and lipids solubilized by a non-ionic detergent Triton X-100 was shown. A removal of the detergent results in a formation of membranes, which are chemically close to the original ones but ultrastructurally very different from the latter. On the other hand, absorption or dialysis of cholate-solubilized proteins and lipids results in reconstituted membranes with asymmetrically arranged intramembrane particles located on the hydrophobic surfaces of the membrane halves. The number and size of these particles are similar to those of the original microsomal membranes.

Animals↗