[Covalently cross-linked, functionally active complex of cholesterol-hydroxylating cytochrome P-450 with adrenodoxin].
Explore the source record for details and available documents.
Adrenal Cortex↗
Biomedical subjects
A A Akhrem
Publications and source records attributed to A A Akhrem.
[Spectrophotometric study of cytochrome P-450 (11 beta) interaction with physiological effectors].
Using the optical absorbance spectroscopy method, the interaction of a number of biospecific ligands (steroids, adrenodoxin) with homogeneous cytochrome P-450 (11 beta) from bovine adrenal mitochondria was investigated. The parameters of the steroid-protein interaction in a number of substrates and products of the 11 beta- and 18 (19)-hydroxylation with the active site of cytochrome P-450 (11 beta) were determined. A sharp decrease in the cytochrome affinity for steroids upon the insertion of the first hydroxy group was observed, which provides for a predominant formation of monohydroxylated products from the substrate and minimum amounts of dihydroxylated ones, despite the presence of more than one position for the substrate hydroxylation by cytochrome P-450 (11 beta). Some structural elements of the steroid molecule were determined as any alterations in these strongly affect the enzyme affinity for the steroid. These structures are: 1) delta 4-3-oxo structure; 2) either 21-hydroxy group of pregnen steroids or the one fulfilling its functions, 17 beta-hydroxy or 17-oxo group of androsten steroids, and 3) the 11th position of all the substrates under study. It was shown that the binding of various substrates into stoichiometric (1:1) steroid-protein complexes provides a transition to high spin state from 30-40% (cortisol, corticosterone) to 90-95% (11-deoxycorticosterone) of hemoprotein iron. Using the experimental system containing individual cytochrome P-450 (11 beta) and adrenodoxin, as well as the steroid and nonionic detergent Tween 20, it was shown that the parameters of substrate binding and hemoprotein spin equilibrium did not differ from the corresponding parameters of the cytochrome-adrenodoxin dienzyme complex. The peculiarities of the multiligand interactions in the 11 beta-hydroxylase system, involving cytochrome, substrates and ferredoxin demonstrate some analogy with a bacterial camphor hydroxylase system and some differences from the mitochondrial system for the side chain cleavage of cholesterol.
Adrenal Glands↗
Domain structure of adrenocortical cytochrome P-450scc: localization of functionally important sites in the polypeptide chain.
Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under its hydrolysis with trypsin the domains retain their associated state due to rigid noncovalent interactions. The structural characteristics of the individual domains have been investigated. It is established that domain I containing the haem and the adrenodoxin-binding site is the N-terminal, and domain II the C-terminal moiety of the polypeptide chain of cytochrome P-450scc.
Adrenal Cortex↗
[Anomalous thermotropic behavior of proteoliposomes consisting of cytochrome P-450, dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol].
Explore the source record for details and available documents.
Biophysical Phenomena↗
[A shunted system of electron transport from NAD(P)H to cholesterol-hydroxylating cytochrome P-450 in adrenal cortex mitochondria].
The two main approaches presently used for cytochrome P-450scc modelling are as follows: i) the use of chemical compounds carrying activated oxygen species, e. g., peracids, organic hydroperoxides, iodosobenzene, etc., ii) the use of electrochemical reduction in the presence of redox-active compounds. In the present work, a new model system for simulation of steroidogenic electron transfer is proposed, which reduces cytochrome P-450 scc by NADPH in the absence of adrenodoxin reductase and adrenodoxin. Phenazine methosulfate is used as an electron carrier. More than 95% of cytochrome P-450scc is reduced in a model system. The reduction kinetics is characterized by a lag phase, thus indicating complex formation between cytochrome P-450scc and phenazine methosulfate or formation of intermediate reducing equivalents. NADH may also serve as an electron donor for cytochrome P-450scc. Phenazine methosulfate can reduce microsomal cytochrome P-450 LM2 and b5, but not cytochrome P-450 LM4. Superoxide dismutase does not affect the reduction, thus indicating that O9.- is not involved in the reduction process. The mechanism of hemoprotein reduction and the nature of intermediates which can be formed in the model system is proposed.
Adrenal Cortex↗
The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. Localization of haem group and domains in the polypeptide chain.
Cytochrome P-450scc consists of two domains linked with a short loop of the polypeptide chain; under hydrolysis by trypsin the domains retain their associated state due to rigid noncovalent interactions. A partial separation of the domains by gel-chromatography on Sephadex G-200 with retention of a haem group in domain I has been achieved after incubation of the trypsin-modified cytochrome P-450scc in 50 mM phosphate buffer (pH 7.2)/1 M NaCl/0.3% sodium cholate/0.3% Tween 80. The separation of domains I and II to individual fragments of the haemoprotein polypeptide chain has been achieved by chromatography under denaturation conditions on the activated thiopropyl-Sepharose via a selective covalent immobilization of domain II. Dissociation of a complex of domains I and II has been effectuated in the presence of 7 M guanidine. Structural characteristics of individual domains have been investigated. It is established that domain I containing a haem group is the N-terminal moiety, and domain II, the C-terminal moiety of the polypeptide chain of cytochrome P-450scc. The pathways of limited trypsinolysis of the native cytochrome P-450scc have been determined. The peptides containing cysteine residues localized on the surface of domain II and responsible for the interaction of haemoprotein with activated thiopropyl-Sepharose have been isolated in a homogeneous form and their amino-acid sequences have been assessed.
Adrenal Cortex↗
Pregnancy-associated molecular variants of human serum transcortin and thyroxine-binding globulin.
Affinity chromatography on immobilised concanavalin A revealed that transcortin and thyroxine-binding globulin isolated from human postpartum serum contained approximately 10% of molecular variants that did not occur in these glycoproteins isolated from normal donor serum (both male and female). The chromatographic behaviour of the pregnancy-associated glycoprotein variants, their monosaccharide compositions, and the results of methylation analysis indicated that these variants contained only triantennary oligosaccharide chains of the N-acetyl-lactosamine type.
Chromatography, Affinity↗
Chemical modification of adrenocortical cytochrome P-450scc with tetranitromethane.
Selective chemical modification of adrenocortical cytochrome P-450scc, responsible for key stages of steroid biogenesis, with tetranitromethane has been carried out. Nitration of the cytochrome P-450scc tyrosine residues results in heme protein inactivation with syncatalytic loss of enzyme activity. Analysis of the cytochrome P-450scc inactivation kinetics indicates that there are several pools of tyrosine residues, differing in their accessibility to tetranitromethane. The modification of cytochrome P-450scc results in changes in the hemeprotein spectral properties and its conformation which indicates to the involvement of essential tyrosine residue(s) in the heme-protein interaction. Cholesterol and adrenodoxin (high-spin effectors) prevent the inactivation of cytochrome P-450scc with tetranitromethane, i.e., protect the essential tyrosine residue(s) from modification. Possible functions of the tyrosine residues in the cytochrome P-450scc molecule are discussed.
Adrenal Cortex↗
The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria.
A homogeneous cytochrome P-450scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregnenolone to be catalyzed by single species of cytochrome P-450scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with Mr 27000 and 22000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, Mr of about 14000. Cytochrome P-450scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.
Adrenal Cortex↗
[Selective chemical modification of cholesterol hydroxylating cytochrome P-450 from adrenal cortex mitochondria by tetranitromethane].
Selective chemical modification of adrenocortical cytochrome P-450 responsible for the key stages in steroid biogenesis has been carried out. Nitration is followed by the heme-protein inactivation and loss of enzymatic activity. Analysis of the cytochrome P-450 nitration kinetics revealed several types of tyrosine residues differing in their accessifility for the reagent. Modification of only one or two tyrosine residues brought about the enzyme inactivation. Modification was accompanied by changes in the cytochrome P-450 spectral properties. Cholesterol and adrenodoxin protected cytochrome P-450 against inactivation by tetranitromethane. The data obtained are discussed in terms of functional role of tyrosine residues in the cytochrome P-450 molecule.
Adrenal Cortex↗
[Paramagnetic effects in the magnetic circular dichroism spectra of cytochromes P-450 LM2 and LM4 from the liver microsomes of rabbits].
Explore the source record for details and available documents.
Animals↗
Selectively immobilized cytochrome c as an effective affinity ligand for electron transfer 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 modification of free carboxyl groups by histamine. Dissociation constants have been determined for complexes of adrenodoxin, hepatoredoxin , cytochrome b5 heme-containing tryptic fragment and myoglobin with cytochrome c preparations immobilized via lysine residues (cytochrome c-Sepharose I) or additional imidazole groups (cytochrome c-Sepharose II). The latter adsorbent possesses a 2-3 times higher affinity to adrenodoxin and hepatoredoxin than the former. The parameters of interaction with cytochrome c-Sepharose II constitute for the proteins studied the following sequence: adrenodoxin (the highest affinity) greater than or equal to hepatoredoxin greater than cytochrome b4 heme- containing tryptic fragment greater than myoglobin. The efficiency of cytochrome c-Sepharose II application in the course of adrenodoxin, hepatoredoxin and cytochrome b5 purification, as well as isolation of cytochrome b4 heme-containing tryptic fragment has been shown.
Adrenal Cortex↗
[Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria].
Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).
Adrenal Cortex↗
[Localization of tetranitromethane-modified tyrosine residues in domains of cholesterol-hydroxylating cytochrome P-450].
As a continuation of earlier structure-function studies on cholesterol-hydroxylating cytochrome P-450 from the adrenal cortex mitochondria, the present study is concerned with the distribution of tetranitromethane-modified tyrosine residues in the hemo-protein domains. With the aid of thiol-disulfide exchange chromatography and SDS polyacrylamide gel electrophoresis the F1 and F2 fragments of the modified cytochrome P-450 were isolated. Nitrated tyrosine residues were found in the F1 fragment, a heme-containing catalytic domain of the molecule.
Adrenal Cortex↗
Effect of the gel-liquid crystalline phase transition on the reduction of cytochrome P-450 reconstituted into dimyristoyl-phosphatidylcholine liposomes.
The isolated purified components of the hepatic endoplasmic monooxygenatic system were incorporated into liposomes (dimyristoyl-phosphatidylcholine) by gel filtration technique. The temperature dependence of the reduction reaction in the range from 11 degrees C to 37 degrees C revealed a break in the Arrhenius-diagram at the phase transition temperature of dimyristoylphosphatidylcholine. The break point is in agreement with thermodynamic parameters derived from microcalorimetric studies of the monooxygenatic system reconstituted in the same way.
Animals↗
[Molecular organization of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria. Chemical modification with bifunctional reagents in the study of oligomeric forms].
The molecular organization of adrenocortical cytochrome P-450scc has been investigated using chemical modification with bifunctional imidates. The oligomeric organization of cytochrome P-450scc in solution has been shown. The application of dimethyl-3,3'-dithiobispropioimidate and subsequent cleavage of the modified products by reducing agents revealed the presence of two types of intramolecular cross-links: "short" at the distance of 3,0 A between the amino groups of lysine residues and "long" ones at a distance of 11,9 A. The analysis of the products, obtained by limited proteolysis of the oligomeric forms of the cross-linked cytochrome P-450, by two-dimensional electrophoresis has shown that the cross-links are formed between the functional domain (fragment F1) and domain responsible for the interaction with the phospholipid membrane (fragment F2). A model for cytochrome P-450scc molecular organization has been suggested on the basis of the obtained results.
Adrenal Cortex↗
[Calculation of stable conformations of the molecule of a serum thymic factor].
Stable conformations of "facteur thimique sérique" (FTS) were determined by semi-empirical conformational analysis. The interpretation of the calculation results in combination with data on biological activity of the conformationally restricted FTS analogues enabled the delineation of a possible "biologically active" conformation.
Amino Acid Sequence↗
[Interaction of 8-aza-16-oxasteroids with rat liver microsomal cytochrome P-450].
The interaction of the 8-aza-16-oxasteroid series (8-AS) with cytochrome P-450 from liver microsomes of intact and phenobarbital-induced rats has been studied. It has been shown that 8-AS are the substrates for the cytochrome P-450-dependent enzyme system, and that their affinity for cytochrome P-450 is determined by the structure of the compounds tested. Using inhibitory analysis, the site in the active center of hemoprotein responsible for 8-AS binding was examined. The possibility of direct participation of the 8-AS ketogroup in their binding with cytochrome P-450 is discussed.
Animals↗