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A A Akhrem

Publications and source records attributed to A A Akhrem.

At least 37 records · Page 2Linked to original sources
PubMed1989

[Interaction of cholesterol hydroxylating cytochrome P-450 with cytochrome b5].

Some new relations between cytochrome P-450-dependent monooxygenases were discovered. Cytochrome b5, a representative of "microsomal" monooxygenases, was shown to form a highly specific complex with cytochrome P-450scc, a member of the "ferredoxin" monooxygenase family. This interaction is characterized by a dissociation constant, Kd, of 0.28 microM. The cytochrome P-450scc-cytochrome b5 complex may be cross-linked with water-soluble carbodiimide. Using proteolytic modification of cytochrome b5, it was shown that both hydrophilic and hydrophobic fragments of cytochrome b5 are involved in the interaction with cytochrome P-450scc. Cytochrome b5 immobilized via amino groups is an effective affinity matrix for cytochrome P-450scc purification. The role of some amino acid residues in cytochrome P-450scc interaction with cytochrome b5 was studied. The role and the nature of complexes in cytochrome P-450-dependent monooxygenases as well as interrelationships between "microsomal" and "ferredoxin" monooxygenases are discussed.

Adrenal Cortex↗
PubMed1988

Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure.

In studying molecular mechanisms of specialised transduction, the lacI (E. coli)-Ea47 (lambda) DNA junction in transducing bacteriophage lambda plac 5 has been structurally elucidated, thus yielding the complete sequence of lambda plac 5 DNA including the lac5 substitution, a well-known segment of lambdoid vectors. The lambda plac5 DNA is shown to consist of 19368 bp (lambda left arm) + 3924 bp (lac5 substitution) + 25353 bp (lambda right arm), totally amounting to 48645 bp. The presence of the phage rho bL promoter near to the right end of the lac5 insert is shown. The lacI gene distal end in lambda plac5 proved to be much longer than it was postulated earlier, coding for 224 C-terminal amino acid residues of lac repressor. Both the recombination studied in this paper and the earlier studied abnormal prophage excision (2, 3) occur near to Chi-like structures (chi*lacI and chi*lom, respectively). On the basis of the data obtained, a key role of the E. coli RecBCD system and Chi-like sequences in the formation of deletions in bacterial cells is suggested.

Bacteriophage lambda↗
PubMed1988

Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.

Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.

Adrenodoxin↗
PubMed1988

Method of modeling protein structure by the two-dimensional nuclear magnetic resonance spectroscopy data; application to the proteinase inhibitor BUSI IIA from bull seminal plasma.

A new approach is suggested to model the spatial structure of protein molecules in solution based on combined use of the methods of theoretical conformational analysis and NMR spectroscopy data. At the first stage, special means are used to convert d connectivity information into the most probable values of dihedral angles. This allows search for possible spatial structures in the limited regions of the conformational space at further stages using the methods of the theoretical conformational analysis. The suggested approach was verified in reconstructing the spatial backbone structure of the fragment 17-57 of the proteinase inhibitor BUSI IIA from the bull seminal plasma. The structural model parameters are compared with the corresponding characteristics obtained from the X-ray analysis data for the homologic proteinase inhibitor from the Japanese quail ovomucoid. The suggested approach is shown to correctly reproduce both the general molecule topology and the conformations of individual amino acid residues.

Amino Acid Sequence↗
PubMed1988

[Mechanism of electron transport in the cholesterol-hydroxylating system of adrenal cortex mitochondria: a triple complex of adrenodoxin reductase, adrenodoxin and cytochrome P-450].

A cross-linked ternary adrenodoxin reductase-adrenodoxin-cytochrome P-450scc complex with an apparent molecular mass of 114 kD was obtained, using N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)-hexanoate. The composition of the cross-linked complex was determined by immunoblotting and radioactivity measurements, using N-ethyl [2.3-14C]maleimide-premodified adrenodoxin. The data obtained suggest that the ternary complex may exist in solution.

Adrenal Cortex↗
PubMed1988

[The covalent-sorption reconstitution of steroid hydroxylases].

The effect of covalent immobilization via free amino groups on the catalytic activity of individual components of the cholesterol side-chain cleavage and 11b-steroid hydroxylation systems (adrenodoxin reductase, adrenodoxin, cytochrome P-450scc and cytochrome P-450(11)b) as well as on that of co-immobilized protein complexes. The protein complex formation at different stages of the monooxygenase cycle (i.e., reduction, oxygenation) was followed by direct spectrophotometric monitoring of the functional state of the immobilized complexes. Cholesterol side-chain cleavage was carried out in minicolumns, using various combinations of immobilized and soluble proteins. Cytochromes P-450scc and P-450(11)b were found to retain their functional activities after immobilization via free SH-groups.

Catalysis↗
PubMed1988

[Isolation and characteristics of coumarin-specific cytochrome P-450 (P-450Cho) from liver microsomes of DBA/2N mice, induced with pyrazole].

Coumarin-specific cytochrome P-450 (P-450Coh) has been isolated from liver microsomes of DBA/2N mice induced with pyrazole. The induction effect was accompanied by a 5.8-5.9-fold increase in the P-450Coh content which made up to 14.4-17% of the total cytochrome P-450 pool in the microsomes. At the final step of P-450Coh purification, variously substituted Sepharoses (hydroxyphenyl-, cholate-, aminooctyl- and t-cytochrome-b5-) were used. The optimal scheme involved solubilization of microsomes with sodium cholate, hydrophobic chromatography on octyl-Sepharose, adsorption on calcium-tartrate gel and hydrophobic ion-exchange chromatography on aminooctyl-Sepharose. According to SDS gel electrophoresis data, the purity of P-450Coh was 95% and Mr was 50,000 Da. The amino acid composition of the protein includes 445 residues. At saturating concentrations of coumarin, more than 90% of P-450Coh are represented by the high spin form. The catalytic activity of P-450Coh was studied in reactions of xenobiotics oxidation.

Animals↗
PubMed1987

Prediction of exposure degree diagram and sites of limited proteolysis in globular proteins as an approach to computer-aided design of protein bioregulators with prolonged action.

In order to prolong the lifetime of protein bioregulators in blood it is possible to engineer analogs with protected sites of limited proteolysis. To determine the sites, primarily accessible to trypsin-like proteases, a computer procedure has been developed, including a prediction algorithm, to produce the residue diagram of a globular protein and a discriminant algorithm to determine the sites most liable to proteolysis. The accuracy of prediction of amino acid residue exposure is characterised by correlation coefficients between experimental and theoretical exposure values, the coefficients being about 0.7 as calculated for 10 globular proteins. The classification of Arg and Lys residues into two groups, susceptible or insusceptible to protease, has an error percentage of about 25.

Arginine↗
PubMed1987

Method of determining protein conformations by the two-dimensional nuclear Overhauser enhancement spectroscopy data.

A method is suggested to determine the most probable values of the angles phi, psi of the protein backbone by the data on the availability and absence of d connectivities in the two-dimensional nuclear Overhauser enhancement spectra. In view of this, the dependences of the proton-proton distances in dipeptide units of L-amino acid residues on the dihedral angles phi, psi, chi1 are considered and the conformational states of amino acid residues of the proteins with the known spatial structure are analysed statistically. The potentialities of the method are assessed with the aid of model spectral nuclear magnetic resonance (NMR) parameters obtained from the X-ray data for the bovine pancreatic trypsin inhibitor and avian pancreatic polypeptide. It is shown that the developed procedure of structural interpretation of the NMR data allows one to correctly reproduce the local conformation of the protein backbone. The obtained backbone conformation may serve as a starting point to build and refine molecular three-dimensional structure.

Aprotinin↗
PubMed1987

[Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450. Monospecific antibodies against domains F1 and F2].

Highly specific antibodies to adrenocortical cytochrome P-450scc as well as fragments F1 and F2 representing the N- and C-terminal sequences of the hemoprotein obtained by limited trypsinolysis were raised in rabbits. Antibodies to cytochrome P-450scc as demonstrated by the Ouchterlony diffusion analysis, immunoelectrophoresis and immunoblotting techniques interact with the hemoprotein and both fragments. Antibodies to cytochrome P-450scc fragments interact with the hemoprotein and corresponding antigens, but do not cross-react. To determine the localization of antigenic determinants in the polypeptide chain of cytochrome P-450scc, the interaction of antibodies to the hemoprotein and to its fragments F1 and F2 with limited trypsinolysis products was studied. All antibodies were found to effectively inhibit cholesterol transformation into pregnenolone in a reconstituted system. Using SDS electrophoresis followed by immunoblotting, the cross-reactivity of antibodies to cytochrome P-450scc and to its fragments with microsomal cytochromes P-450scc LM2 and LM4 as well as with mitochondrial cytochrome P-45027 was revealed. This finding testifies to the presence of common antigenic determinants in the hemoproteins.

Adrenal Cortex↗
PubMed1987

[Fatty-acid initiated auto-oxidation of hemoglobin].

The interaction of fatty acids (FA) with oxyhemoglobin (HbO2) was investigated. It was found that under effect of FA HbO2 is converted into the oxidized low-spin form, i. e., hemichrome. An increase in the level of reactive oxygen species was registered by the ESP method, using the spin traps, 1.2-dioxybenzo-3.5-disulphonate (tiron) and 5.5-dimethylpyrroline-1-oxide (DMPO). To investigate the nature of radical particles, the OH radical traps. thiourea and D-mannitol, as well as superoxide dismutase (SOD) and catalase were employed. The mechanism of interaction of HbO2 and FA is discussed; its feasible physiological role in the functioning of erythrocytes is postulated.

Animals↗
PubMed1987

[Chemical modification of cholesterol-hydroxylating cytochrome P450 from bovine adrenal cortex mitochondria by acetylimidazole].

The state and role of tyrosine residues in adrenocortical cytochrome P-450scc was investigated. Spectrophotometric titration experiments showed the existence of two types of tyrosine residues in the hemoprotein molecule, i.e., exposed (pK 9.25) and buried (pK 10.75) ones. Chemical modification of the exposed tyrosine residues with N-acetylimidazole was carried out. The data obtained indicate the involvement of these residues in the interaction with adrenodoxin.

Adrenal Cortex↗
PubMed1987

[Effector role of fatty acids in the transformation of methemoglobin into hemichrome].

The interaction of fatty acids (FA) with methemoglobin (met-Hb) was investigated. It was found that under the action of FA met-Hb is rapidly converted into a low-spin form (the so-called hemichrome). This process is reversible as proved by the addition of bovine serum albumin into the system. Using differential spectrophotometry, the efficiency of FA action on met-Hb depending on concentration and nature of FA was estimated. Using the circular dichroism (CD) method, it was demonstrated that under the action of FA the concentration of R-conformation in the met-Hb molecule is increased. The stoichiometry of the hemichrome complex with FA at various FA concentrations was assessed. A comparative analysis of the effect of various detergents with known structures on met-Hb was carried out. The mechanism of met-Hb conversion into hemichrome is discussed.

Animals↗
PubMed1987

[Comparative study of the C27-steroid-hydroxylating system from bovine liver mitochondria].

A method for purification of C27-steroid hydroxylating cytochrome P-450 (cytochrome P-450(27)) from bovine liver mitochondria was developed. The purification procedure included enzyme extraction from submitochondrial particles with sodium cholate, ammonium sulfate fractionation and biospecific chromatography on cholate-Sepharose and adrenodoxin-Sepharose. The resulting enzyme preparation (317-fold purification, 16% yield) was not electrophoretically homogeneous but did not contain hemoprotein admixtures. The kinetic parameters of 5 beta-cholestane-3 alpha,7 alpha,12 alpha-triol 27-hydroxylation in a reconstituted system containing hepatoredoxin reductase, hepatoredoxin and cytochrome P-450(27) (Km = 23 microM, kcat = 0.3 s-1 at 25 degrees C) were determined. A reciprocal functional equivalency of hepatoredoxin reductase and adrenodoxin reductase as well as of hepatoredoxin and adrenodoxin in reconstituted systems of steroid 27-hydroxylation (liver) and cholesterol side chain cleavage (adrenal cortex) was established. This equivalency was thought to be due to the similarity in essential physico-chemical properties of reductase components which was especially well-pronounced in the case of hepatoredoxin and adrenodoxin. Estimation of the functional role of lysine, dicarboxylic acid and histidine residues in ferredoxin molecules by the chemical modification method revealed the similarity of the structural organization of their protein globules: the polar residues were shown to be essential for the maintenance of native conformation; dicarboxylic acid residues formed a binding domain for the interaction with electron transport proteins, whereas histidine residues seem to participate in electron transport. At the same time, cytochrome P-450(27) and cytochrome P-450 which split the side chain of cholesterol differ in their substrate specificity, immunochemical and catalytic properties.

Adrenal Cortex↗