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Biomedical subjects

A A Akhrem

Publications and source records attributed to A A Akhrem.

At least 55 records · Page 3Linked to original sources
PubMed1987

[Kinetics of 15 alpha-methyl-8-aza-16-oxagona-1,3,5(10), 13-tetraen-17-ona oxidation by cytochrome P-450 from rat liver microsomes].

The kinetics of oxidation of 15 alpha-methyl-8-aza-16-oxagona-1,3,5(10),13-tetraen-17-on with rat liver microsomal cytochrome P-450 was investigated. The kinetic parameters, Km and Vmax, of the oxidation reaction were found to be equal to 1,3 X 10(-4) M and 4.0 X 10(-7) M X s-1, respectively. Using thin-layer chromatography, mass-spectrometry, PMR-spectroscopy and reciprocal synthesis, it was shown that 3-hydroxy-15 alpha-methyl-8-aza-16-oxagona-1,3,5(10), 13-tetraen-17-on is the main reaction product.

Animals↗
PubMed1987

[Chemical modification of cysteine residues of cholesterol-hydroxylating cytochrome P-450. Identification of the cysteine residue participating in the formation of a proximal ligand].

A chemical modification of cysteine residues in mitochondrial cytochrome P-450scc from adrenal cortex has been carried out. Cysteine residues in this hemoprotein were shown to form two pools: one available to the chemical modification, which does not affect spectral and functional properties of the cytochrome and another accessible for the modification only after the protein inactivation and the heme removal. The proximal ligand in the polypeptide chain of cytochrome P-450scc was localized. Cys422 was shown to be involved in the heme coordination and Cys264 was found to be exposed and accessible to sulfhydryl reagents. The data obtained are discussed in terms of the functional role of cysteine residues in monooxygenase catalysis.

Adrenal Cortex↗
PubMed1987

[Localization of tetranitromethane-modified tyrosine residues in the polypeptide chain of cholesterol-hydroxylating cytochrome P-450].

As a continuation of earlier structure-function relationship studies on cholesterol-hydroxylating cytochrome P-450 from the adrenal cortex mitochondria, the present study deals with the distribution of tetranitromethane-modified tyrosine residues in the hemeprotein polypeptide chain. Amino acid residues Tyr-24, -46, -50, -93, -94, -199, -246 are shown to be modified with tetranitromethane. Tyr-93, -94 are supposedly involved in the active site formation of cytochrome P-450.

Adrenal Cortex↗
PubMed1986

Primary structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria and some aspects of its functioning on a structural level.

The primary structure of the cholesterol side-chain cleavage cytochrome P-450 (P-450scc) from bovine adrenocortical mitochondria has been determined. At the initial stage an exhaustive chymotryptic digestion of carboxymethylated P-450scc was performed, and the amino acid sequence of 66 peptides was determined. At the second stage an investigation of the amino acid sequence of individual fragments I (Mr 29 800) and II (Mr 26 600) of the limited trypsinolysis of P-450scc was carried out. Fragment I was digested with trypsin, Staphylococcus aureus V8 proteinase and thermolysin; fragment II was cleaved with trypsin and S. aureus V8 proteinase. In addition, the amino acid sequence of some CNBr peptides of P-450scc has been investigated. The primary structure of cytochrome P-450scc determined with protein chemistry methods proved the multistage cholesterol transformation to pregnenolone to be catalyzed by a single species of cytochrome P-450scc which consists of 481 amino acids. The results from protein sequencing of P-450scc are in good agreement with those obtained recently from nucleotide sequencing. The localization of peptide bonds cleaved under limited proteolysis of P-450 with trypsin to fragments I and II, I and III (Mr 16 800) is presented. It is shown that the transformation of P-450scc to P-420 is accompanied by the appearance of an additional trypsin-sensitive peptide bond in the N-terminal part of P-450scc.

Adrenal Cortex↗
PubMed1986

[Complete primary structure of DNA from the transducing bacteriophage lambda plac5].

In studying molecular mechanisms of specialized transduction, primary structure of the junction between the E. coli gene lacI and the lambda phage locus Ea47 in transducing bacteriophage lambda plac5 has been established. Along with the lambda DNA and E. coli lac operon structures as well as with our earlier data on another phage-bacterial junction in lambda plac5, it lead to the complete sequence of lambda plac5 DNA, including the lac5 substitution, a wellknown segment of lambdoid cloning vehicles. The lambda plac5 DNA is shown to consist of 48645 b.p. distributed as follows: 19368 (lambda left arm) + 3924 (lac5 substitution) + 25353 (lambda right arm). The presence of the phage pbL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be considerably more long-stretched than it used to be believed, coding for 224 C-terminal amino-acid residues of lac repressor. The recombination studied in this paper, similarly to the abnormal prophage excision, occurred near to a Chi-like structure, which is partly homologous to the chi+lacZ site present in lambda plac5. On the basis of the data obtained, a key role of the E. coli RecBC system and Chi sites in the formation of long-stretched deletions in the bacterial cell has been suggested.

Bacteriophage lambda↗
PubMed1986

[Molecular organization of the reductase complex in adrenal cortex mitochondria. Study using bifunctional reagents].

The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, homobifunctional reagent 3,3'-dithiobis (succinimidyl propionate), and heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio) propionate have been used to cross-link adrenodoxin reductase and adrenodoxin, components of steroidogenic electron transfer system. Though maximal yield of the cross-linked complex was achieved with the water-soluble carbodiimide, this complex was inactive in the electron transfer from NADPH to cytochrome P-450. The functionally active complex was formed with N-succinimidyl 3-(2-pyridyldithio) propionate. The complex was purified to the apparent homogeneity and shown to be able to mediate the electron transfer. The data obtained indicate existence of different binding sites on adrenodoxin responsible for the adrenodoxin reductase and cytochrome P-450scc binding and do not contradict to the model of the steroidogenic electron transfer in an organized complex.

Adrenal Cortex↗
PubMed1985

Cross-linking studies of steroidogenic electron transfer: covalent complex of adrenodoxin reductase with adrenodoxin.

Bifunctional reagents 3,3'-dithiobis(succinimidyl propionate), 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide and N-succinimidyl 3-(2-pyridyldithio)propionate have been used in an attempt to study molecular organization and covalent cross-linking of adrenodoxin reductase with adrenodoxin, the components of steroidogenic electron transfer system in bovine adrenocortical mitochondria. There was no cross-linking of individual proteins by the bifunctional reagents used, except for adrenodoxin cross-linking with water-soluble carbodiimide. Substantial cross-linking of adrenodoxin reductase with adrenodoxin was observed when water-soluble carbodiimide was used as cross-linking reagent. However, the cross-linked complex failed to transfer electrons. Significant amounts of the functional cross-linked complex (up to 42%) were observed when the proteins were cross-linked with N-succinimidyl 3-(2-pyridyldithio)propionate. Using gel filtration, ion-exchange chromatography and affinity chromatography on adrenodoxin-Sepharose, the complex was obtained in a highly purified form. In the presence of cytochrome P-450scc or cytochrome c, the cross-linked complex of adrenodoxin reductase with adrenodoxin was active in electron transfer from NADPH to heme proteins. The data obtained indicate that there are distinct binding sites on the adrenodoxin molecule responsible for the adrenodoxin reductase and cytochrome P-450scc binding, which suggests that steroidogenic electron transfer may be realized in an organized complex.

Adrenal Cortex↗
PubMed1985

The inhibition of mitochondrial F1-ATPase by 1,5-difluoro-2,4-dinitrobenzene.

1,5-Difluoro-2,4-dinitrobenzene completely inhibits F1-ATPase when it is used at micromolar concentrations and the inhibitor/enzyme molar ratio is equal to 3. The inhibition can be reversed by dithiothreitol treatment. 7-Chloro-4-nitrobenzofurazan treatment of F1-ATPase does not prevent the reaction of the enzyme with 1,5-difluoro-2,4-dinitrobenzene. The 1,5-difluoro-2,4-dinitrobenzene-induced inhibition is thought to be a result of the modification of a tyrosine residue with pK 9.1.

4-Chloro-7-nitrobenzofurazan↗
PubMed1985

Cross-linking studies of adrenocortical cytochrome P-450scc. Evidence for a covalent complex with adrenodoxin and localization of the adrenodoxin-binding domain.

A cleavable cross-linking reagent, dimethyl-3,3'-dithiobispropionimidate, was used to study the molecular organization of adrenocortical cytochrome P-450scc. Extensive cross-linking was found to occur, resulting in the formation of heterologous oligomers up to octamer. The covalently cross-linked complex of adrenocortical cytochrome P-450scc with adrenodoxin has been obtained by using dimethyl-3,3'-dithiobispropionimidate. In the presence of NADPH and adrenodoxin reductase, electron transfer to cytochrome P-450scc occurs in the complex, and, in the presence of cholesterol, the latter effectively oxidizes to pregnenolone. By using covalently immobilized adrenodoxin and heterobifunctional reagent, N-succinimidyl-3-(2-pyridyldithio)propionate, the adrenodoxin-binding site was shown to be located in the heme-containing, catalytic domain of cytochrome P-450scc. The data obtained indicate the existence of two different sites on the adrenodoxin molecule that are responsible for the interaction with adrenodoxin reductase and cytochrome P-450scc. This is consistent with the model mechanism of electron transfer in the organized complex.

Adrenal Cortex↗
PubMed1985

[Effect of phosphatidylglycerol on the incorporation process of cytochrome P-450 in liposomes from dimyristoylphosphatidylcholine].

Using the electron paramagnetic resonance (EPR) method with spin-labeled fatty acids and gel-penetrating chromatography, the effect of phosphatidylglycerol on cytochrome P-450 incorporation into liposomes from dimyristoylphosphatidylcholine was investigated. An addition of phosphatidylglycerol caused an increase in the protein content of the proteoliposomes as well as their accelerated formation at temperatures below and above the liposome phase transition temperature (Ts). The dependence of the proteoliposome formation rate on the phosphatidylglycerol content in the liposome mixture is described by complex kinetics with a maximum in the presence of 10 mol.% of the negatively charged phospholipid. The mechanism of proteoliposome formation is discussed in terms of asymmetric distribution and phase state of the phospholipids in the original vesicles.

Animals↗
PubMed1985

Chemiluminescence of luminol caused by interaction of cytochrome P-450 and cytochrome C with cumene hydroperoxide: comparative studies.

A comparative study of the interaction of cumene hydroperoxide with cytochrome P-450LM2 and with cytochrome C has been undertaken using the chemiluminescence method in the presence of luminol. Considerable differences in the mechanisms of action of both hemoproteins have been revealed with various inhibitors of radical stages, i.e. superoxide dismutase, mannitol, sodium azide, and alpha-tocopherol. It is shown that molecular oxygen participates in the process of hemoprotein-catalyzed hydroperoxide oxidation of luminol.

Animals↗
PubMed1985

[Reconstruction and study of a multi-enzyme system by 11 beta-hydroxylase steroids].

The reconstitution of the steroid 11 beta-hydroxylase system based on the homogeneous proteins isolated from bovine adrenocortical mitochondria, cytochrome P-450 (P-450 (11 beta), 19-20.5 nmol of heme P-450 per 1 mg of protein), adrenodoxin (Adx) and adrenodoxin reductase (AR) was carried out. The reconstitution of the multienzyme system requires the presence of a non-ionic detergent due to the high hydrophobicity of P-450 (11 beta). Low concentrations of Tween 20 (below 0.015% or 115 microM) stimulate the reaction of steroid 11 beta-hydroxylation by improving the hemoprotein solubility. With a further increase in the detergent concentration, the reaction is inhibited due to the inactivation of the cytochrome and its impaired interaction with Adx. The electron transfer activity of adrenodoxin reductase and the dienzyme AR-Adx complex does not change within the Tween 20 concentration range of 0-0.4%. In solutions with the optimal concentration of Tween 20 (0.010-0.015%), the concentrations of AR and Adx providing for the half-maximum hydroxylation activity are 9 nM for AR and 280 nM for Adx. It was shown that in a reconstituted 11 beta-hydroxylase system, 75% of the reducing equivalents are involved in the formation of oxygen radicals, whereas 25%--in hydroxylation. 74% of the radical species are, in their turn, formed in the active site of the hemoprotein, while 26%--in the Fe2S2 center of adrenodoxin. The radical formation process predominates over the 11 beta-hydroxylation within a wide range of Adx/cytochrome ratios, i.e., 1.0-100. The hydroxylation substrate induces a 4-fold increase in the electron transfer rate by stimulating the enzymatic reduction of P-450 (11 beta), but only 35% of the additional reduced equivalents are consumed by the 11 beta-hydroxylation and 65%--by the oxygen radical formation.

Adrenal Glands↗
PubMed1985

[Various properties of purified hepatoredoxin from bovine liver mitochondria].

Hepatoredoxin purified to homogeneity from bovine liver mitochondria has been characterized for the first time in terms of its most important physico-chemical properties. The protein was found to contain in its active center a [2Fe-2S] cluster and has in the oxidized state an absorption maxima at 280, 320, 415 and 455 nm. The spectrophotometric index of purity, A415/A280 of the homogeneous native preparation is 0.84; extinction coefficient, epsilon 415, is 9800 M-1cm-1. The Mr of hepatoredoxin as evidenced by data from SDS gel electrophoresis is 12 500 Da; pI is 4.2. Hepatoredoxin is necessary for the reconstitution of the C27-steroid hydroxylase activity and can be substituted for by a related protein, adrenodoxin. All the above parameters as well as the circular dichroism spectra, immunochemical properties and sequence of the initial five N-terminal amino acids of hepatoredoxin and adrenodoxin are either coincident or very close. At the same time, the amino acid composition of these ferredoxins, apart from some common features, has individual peculiarities.

Adrenal Cortex↗
PubMed1985

[Effect of chemical modification of tyrosine residues of cholesterol-hydroxylating cytochrome P-450 on the interaction with high-spin effectors].

The role of tyrosine residues of cytochrome P-450scc in the interaction with adrenodoxin and cholesterol was investigated, using chemical modifications with tetranitromethane. Selective chemical modification of tyrosine residues resulted in hemoprotein inactivation. Nitration changed the kinetic parameters of the cholesterol side chain cleavage reaction. Both high spin effectors, i.e., substrate and adrenodoxin, prevent cytochrome P-450scc from the inactivation caused by tetranitromethane. Modification of cytochrome P-450scc decreased the affinity of the hemoprotein for cholesterol and adrenodoxin. Some direct evidence for tyrosine involvement in the interaction between cytochrome P-450scc and adrenodoxin was obtained through the use of zonal affinity chromatography on immobilized ferredoxin.

Adrenal Cortex↗