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Biomedical subjects

A A Akhrem

Publications and source records attributed to A A Akhrem.

At least 91 records · Page 5Linked to original sources
PubMed1984

Reconstitution of the liver microsomal monooxygenase system in liposomes from dimyristoylphosphatidylcholine.

Different techniques to incorporate the essential isolated and purified enzymes of the hepatic endoplasmic reticulum into monolamellar dimyristoylphosphatidylcholine vesicles are compared with respect to structural and functional parameters of the reconstituted system. By use of gel penetrating chromatography on Sepharose 4B, dynamic light scattering and electron microscopy the structural properties of the reconstituted system were proved comparing the reduction of P-450 LM2 via the NADPH dependent reductase and by dithionite as well with the microsomal reduction rates and by studying the binding of benzphetamine to P-450 LM2 in soluble and liposomal form.

Animals↗
PubMed1983

Low-energy Mg2+-induced temperature transitions in liver microsomes.

The differential scanning microcalorimetry and fluorescence methods, using probes ANS and pyrene, have been employed to study thermotropic behaviour of rat liver microsomes in the presence and absence of Mg2+. Addition of Mg2+ yields three partially reversible phase transitions at 18, 27 and 32 degrees C, respectively. A character of Mg2+-induced rearrangements in a membrane and their relation to a catalytic function of a cytochrome P-450-dependent enzymatic system is discussed.

Animals↗
PubMed1983

Study of the carbohydrate moiety of human serum sex hormone-binding globulin.

Sex hormone-binding globulin from human blood serum contains two biantennary N-linked oligosaccharide chains of the N-acetyllactosamine type and one O-linked oligosaccharide per one molecule of the glycoprotein. These conclusions have been based on the results of methylation analysis of the whole glycoprotein and investigation of the structures of its glycopeptides prepared using pronase digestion.

Amino Acids↗
PubMed1983

[The C27-steroid hydroxylating system from bovine liver mitochondria. Isolation of ferredoxin (hepatoredoxin) by affinity chromatography on cytochrome-c-sepharose].

A technique for the isolation of highly purified hepatoredoxin involving the DE-32 cellulose chromatography of post-mitochondrial supernatant, ammonium-sulfate fractionation, Sephadex G-75 gel chromatography, 1-amino-2-hydroxypropyl-Sepharose ion-exchange chromatography and cytochrome-c-Sepharose affinity chromatography is described. The protein was purified 160-fold with a yield of 19%. The synthesis of cytochrome-c-Sepharose was carried out in a way preventing modification of the lysine-containing binding domain of the cytochrome c molecule. To achieve this, free carboxyl groups were modified with histamine to introduce imidazole residues in cytochrome c and the modified protein was immobilized on bromoacetyl-Sepharose.

Animals↗
PubMed1983

[Immobilized cytochrome c--an effective ligand for affinity chromatography of electron transport proteins].

Preparations of horse heart cytochrome c have been obtained immobilized on Sepharose derivatives via lysine epsilon-amino groups, carboxyl groups of aspartic and glutamic acid residues, methionine and histidine residues as well as imidazole groups additionally introduced by means of chemical modification of free carboxyl groups by histamine. Dissociation constants have been determined for complexes of adrenodoxin, hepatoredoxin, cytochrome b5 heme-containing fragment and myoglobin with preparations of cytochrome c immobilized via lysine residues (adsorbent I) or additionally introduced imidazole groups (adsorbent II). The latter is found to possess a 2-3 times greater affinity for adrenodoxin and hepatoredoxin than the former. The affinity of the proteins studied for the adsorbent II constitutes the following sequence: adrenodoxin greater than or equal to hepatoredoxin greater than cytochrome b5 heme-containing fragment greater than myoglobin. The adsorbent II is shown to be effective when used for purification of hepatoredoxin, adrenodoxin, cytochrome b5 and isolation of cytochrome b5 heme-containing fragment.

Adrenal Cortex↗
PubMed1983

[Structural organization of adrenodoxin using limited proteolysis].

The microheterogeneity of adrenodoxin preparation was established by endogenous proteolysis. The controlled limited trypsinolysis and endogenous proteolysis result in modification of the COOH-terminus of the polypeptide chain with a formation of a protein with Mr = 10 000. The interaction of this protein and of the native protein with cholesterol-specific cytochrome P-450 and adrenodoxin reductase occurs in a similar way.

Adrenal Cortex↗
PubMed1982

Thermotropic behaviour of phospholipid vesicles reconstituted with rat liver microsomal cytochrome P-450.

The interaction of rat liver microsomal cytochrome P-450 with phospholipids has been investigated employing differential scanning microcalorimetry. It is shown that the thermotropic behaviour of phospholipid vesicles reconstituted with cytochrome P-450 depends on liposome composition, protein concentration and the mode of reconstituted system preparation. From the delta H dependence on protein concentration in proteoliposomes it was calculated that one cytochrome P-450 molecule influence 350 +/- 50 dimyristoylphosphatidylcholine (DMPC) molecules. The electrostatic interaction of cytochrome P-450 and negatively charged phospholipid, phosphatidylinositol (PI), mixed with DMPC involves the temperature stabilization of proteoliposomes at a phase transition of phospholipid bilayers. The thermal denaturation temperature is increased due to negatively charged PI added.

Animals↗
PubMed1982

Isolation and characterization of glycopeptides of human transcortin.

Pronase digestion of human transcortin yielded two major glycopeptides (GID3 and G2D2, in a molar ratio of approx. 1.0 to 1.5) which were isolated by gel and anion-exchange chromatography. Chromatography behaviour, methylation analysis and analytical chromatography on a Con A-Sepharose column suggested that both of the glycopeptides were N-linked asparaginyl oligosaccharides of the N-acetyllactosamine type, G1D3 and G2D2 being triantennary and biantennary isoglycans, respectively. Microheterogeneity in regard to the position of the linkages between sialyl and galactosyl residues occurred in the triantennary and, possibly, in biantennary isoglycans.

Amino Acids↗
PubMed1982

Structural organization of the carbohydrate moiety of human transcortin as determined by methylation analysis of the whole glycoprotein.

Methylation analysis of human transcortin showed that this glycoprotein contains N-glycosidically linked oligosaccharide chains of N-acetyllactosamine type, most of the chains being biantennary and others tri- and/or tetra-antennary. The carbohydrate chains of transcortin are also heterogeneous with respect to the content of fucose and the position of the glycosidic linkages.

Carbohydrate Sequence↗
PubMed1982

[11-beta-Steroid hydroxylating cytochrome P-450 from bovine adrenal cortex mitochondria: isolation, physico-chemical and functional properties].

It was shown that of the two cytochromes P-450, i.e. cholesterol-hydroxylating (SCC) and 11 beta-steroid-hydroxylating (11 beta) ones, involved in mitochondrial steroidogenesis in adrenal cortex cells cytochrome P-450 (11 beta) is more tightly bound to the membrane. The quantitative ratio of P-450 (SCC)/P-450 (11 beta) in the mitochondrial membrane is 2 to 1. The isolation procedure for cytochrome P-450 (11 beta) included separation of cytochromes P-450 (11 beta) and P-450 (SCC) by ammonium sulfate fractionation in the presence of sodium cholate, Tween 80 and affinity chromatography on adrenodoxin-Sepharose allowing to achieve a high degree of cytochrome P-450 (11 beta) purification. The obtained protein has a high specific heme content (19 nmole per mg of protein), minimal molecular weight (47000), is homogeneous as can be evidenced by Na-DS polyacrylamide gel electrophoresis and N-terminal analysis, possesses spectral properties typical for a high spin hemoprotein and is highly active in the reaction of steroid substrate hydroxylation. The Km values for hydroxylation of deoxycorticosterone (DOC), 11-deoxycorticol and testosterone are 0.69, 0.54 and 0.21 s-1, respectively. The use of adrenodoxin possessing a high stabilizing action allows to obtain cytochrome P-450 (11 beta) free from substrate (a low spin form) by washing of adrenodoxin-Sepharose adsorbed hemoprotein with large volumes of steroid-free phosphate buffer. The eluted cytochrome P-450 (11 beta) reveals spectral properties typical for the low spin hemoprotein; an addition of DOC induces type I spectral changes. The Kd values for the cytochrome P-450 (11 beta)-steroid complex determined by spectrophotometric titration at 10 degrees for DOC and testosterone are 0.32 and 0.67 muM, respectively.

Adrenal Cortex↗
PubMed1982

[Analysis of protein-protein interactions in an enzymatically complete cholesterol hydroxylating system].

The interaction of the cholesterol side chain cleavage system components--adrenodoxin reductase, adrenodoxin, and cytochrome P-450scc was studied, using enzymatic conversion of the high spin form of cytochrome P-450scc to its low spin form and spectrophotometric analysis of various stoichiometric protein mixtures in the presence of pregnenolone and Tween 20. Upon reconstitution of the enzymatically complete system in the presence of NADPH the formation of a ternary protein complex was accompanied by a 2-10-fold increase of the Kd values as compared to the formation of binary adrenodoxin reductase . adrenodoxin and cytochrome P-450 . adrenodoxin complexes. The non-ionic detergent Tween 20 destabilized the adrenodoxin cytochrome P-450scc complex; as a result adrenodoxin reductase acquired the ability to replace adrenodoxin from the binary complex. At the same time this replacement was not observed in the presence of pregnenolone which does not change adrenodoxin affinity for cytochrome P-450scc thus indicating the formation of a ternary protein complex. Titration by adrenodoxin of different stoichiometric mixtures of adrenodoxin reductase and cytochrome P-450scc in the presence of Tween 20 demonstrated that the predominant formation of the binary adrenodoxin . adrenodoxin reductase complex is correlated with an increase of Kd for the cytochrome P-450scc . adrenodoxin complex and that at relatively low detergent concentrations adrenodoxin can interact both with adrenodoxin reductase and cytochrome P-450scc to form a ternary protein complex.

Adrenodoxin↗
PubMed1982

Cumene hydroperoxide supported demethylation of N,N-dimethylaniline by cytochrome P-450 from adrenal cortex mitochondria.

The interaction of highly purified cytochrome P-450 from bovine adrenal cortex mitochondria (cytochrome P-450scc) with N,N-dimethylaniline (DMA), aniline, N-dimethylcyclohexylamine and cumene hydroperoxide (CHP) has been investigated. The formation of complexes between cytochrome P-450scc and the above listed compounds could be demonstrated. The reaction of oxidative demethylation of DMA by cumene hydroperoxide involving cytochrome P-450scc has been carried out at 37 degrees C; the mechanism of this process is discussed. Incubation of cytochrome P-450scc with negatively charged phospholipids, phosphatidylglycerol (PG), and phosphatidylinosite (PI) exerts an inhibiting effect on the reaction of oxidative demethylation. The interaction of cytochrome P-450scc with CHP is accompanied by hemoprotein destruction in a complex biphasic way. The process of oxidative demethylation of DMA in the system of cytochrome P-450scc-CHP has been concluded to have a predominantly radical character.

Adrenal Cortex↗
PubMed1981

[Effects of phospholipids on oxidative demethylation of dimethylcyclohexylamine by cumene hydroperoxide involving methemoglobin].

The reaction of oxidative demethylation of N-dimethylcyclohexylamine by cumene hydroperoxide involving methemoglobin was studied. Data from differential spectroscopy and kinetic analysis revealed the formation of a methemoglobin--N-dimethylcyclohexylamine--cumene hydroperoxide complex. The inhibiting analysis revealed the radical stages in the process of demethylation. An addition to the reaction mixture of phosphatidyl serine, phosphatidyl inositol and lysophosphatidyl choline at a ratio of 50 divided by 500 molecules per 1 molecule of protein increased the rate of the reaction product accumulation 2--3-fold. Phosphatidyl choline and the ionic detergent sodium cholate did not practically affect the reaction rate under the given experimental conditions. The nature of the activating effect of some phospholipids on oxidative demethylation is discussed.

Animals↗
PubMed1980

[Influence of ligands characteristic of selective binding on DNA helix-coil transition. II. Expansion of DNA melting curves into constitutents. Inverse problem of melting of the block heterogeneity DNA. Determination of the content ratio of binding centers to DNA degree of helicity].

The method for determination of distribution density function of base pairs at blocks with different GC-content in DNA (p(kappa)) was developed. For this purpose a differential melting curve is expanded into constituents possessing Gaussian form. Dependences of the content ratio of DNA ligand binding centres of different kinds at helical and coil regions were expressed through p(kappa). These dependences are used for calculation of the ligand influence on a helix--coil transition. It is shown that in spite of incorrectness of p(kappa) determination, the problem of determining content ratio of different kind binding centres expressed through p(kappa) is a correct problem.

Base Composition↗