[Effect of the structure of the brassinosteroid side chain on monooxygenase activity of liver microsomes].

Possible pathways by which brassinosteroids affect the monooxygenase enzymatic system of mammalian liver microsomes, which is involved in the transformation of a broad spectrum of xenobiotics, were studied. The role of the structure of the side chain of brassinosteroids in the regulation of monooxygenase activity was studied using two natural compounds (24-epibrassinolide and 28-homobrassinolide) and two synthetic analogues, (22S, 23S-dihydroxy) stereoisomers. The results of this study show that brassinosteroids can directly influence the functioning of the microsomal enzymatic system. It was found that the degree of this influence depends on the side chain structure. This suggests the possibility of targeted modification of natural compounds to ensure the desired physiological effects.

[Bromoacyl analogues of phosphatidylcholine with intramolecular fluorescence quenching and their use as substrates for continuous monitoring of phospholipase A2 activity]. / Bromatsil'nye analogi fosfatidilkholina s vnutrimolekuliarno potushennoi fluorestsentsiei v kachestve substratov dlia nepreryvnoi registratsii aktivnosti fosfolipazy A2.

Two new derivatives of phosphatidylcholine with intramolecular fluorescence quenching were obtained by substituting residues of pyrene butyric and bromine-containing fatty acids for acyl chains. The two compounds can be used for quantitative evaluation of catalytic activity of pancreatic phospholipase A2 in kinetic mode.

[Comparative study of the effect of fatty acids and certain spin effectors on quaternary structure and spin state of methemoglobin]. / Sravnitel'noe issledovanie vliianiia zhirnoi kisloty i nekotorykh spinovykh éffektorov na chetvertichnuiu struktury i spinovoe sostoianie metgemoglobina.

Cysteine beta-93 SH-groups of horse and bovine methemoglobins (metHb) were modified by N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodoacetamide. Effects of pH, F-, CN-, oleic acid, and sodium dodecyl sulfate on spin and conformational dynamics of the protein were studied by electron and EPR spectroscopy. Oleic acid and CN- had similar effects on both parameters. Relationship between spin and conformational transformations of metHb was elucidated. Effect of oleic acid can be due to increased conformational mobility of C-terminus of metHb beta-chains.

[High performance liquid chromatography of fluorescent insulin derivatives]. / Vysokoéffektivnaia zhidkostnaia khromatografiia fluorestsentnykh proizvodnykh insulina.

The reversed-phase HPLC of porcine insulin modified by fluorescent labeling with dansyl chloride, fluorescein isothiocyanate, and an N-hydroxysuccinimide ester of 3-carboxyl derivative of Nile Red was studied. Mono-, di-, and tri-Dns-insulins (substituted at residues Gly1 of the A-chain and Phe1 and Lys29 of the B-chain), as well as isomeric 5'- and 6'-fluorescein thiocarbamoyl-Phe1 insulin derivatives were separated on the analytical and semipreparative scale. The results were interpreted in terms of conservation of the globular structure in the modified proteins and their surface-mediated interaction with the reversed-phase sorbent. Observed retention times correlated with the total hydrophobicity of the surface region containing the incorporated label (in the case of monosubstituted derivatives) or with the total hydrophobicity of chromatographic contact regions located between labels (in the case of di- and trisubstituted derivatives).

[Structural changes of lipid and thyroxine-binding protein zones of lipoprotein particles, containing human apolipoprotein A-1]. / Strukturnye izmeneniia lipidnoi tiroksinsviazyvaiushchei belkovoi zon lipoproteinovykh chastits, soderzhashchikh apolipoprotein A-1 cheloveka.

The relationship between temperature-induced structural changes in the lipid and protein thyroxine-binding zones of lipoprotein particles containing human apolipoprotein A-1 (apoA-1) as a sole protein component was studied using ESR with probes of two types--spin-labeled thyroxine and 5- or 16-doxylstearic acids. A structural rearrangement in the protein active site at 23-24 degrees was found. In the same temperature range, a dramatic change in the monolayer phospholipid ordered structure was observed. Based on these data, the localization of the apoA-1 thyroxine-binding site in the lipid phase as well as the possibility of regulation of the hormone binding process by changes in the protein-lipid interaction in lipoprotein particles are discussed.

[Isolation, identification and properties of a new thyroxine-binding protein--apolipoprotein A-1 from human blood serum]. / Vydelenie, identifikatsiia i svoistva novogo tiroksinsviazyvaiushchegobelka--apolipoproteina A-1 syvorotki krovi cheloveka.

A protein having a molecular mass of about 25 kWa was isolated by thyroxin (T4)-Sepharose affinity chromatography from human blood serum; its properties were found to be distinct from those of known T4-binding proteins. Using immunodiffusion, radioimmunoassay, lipid analysis, differential precipitation and electrophoresis, it was shown that the isolated protein is a component of high density lipoprotein (HDL) particles and represents an apolipoprotein A-1 (apoA-1). Using cholate-Sepharose chromatography apoA-1 was separated from the lipid moiety and contaminant proteins, and apoA-1 was effectively isolated directly from the blood serum. Apo-A-1-HDL and apoA-1 obtained by affinity chromatography as well as the HDL3 fraction isolated by a standard ultracentrifugation technique, all displayed a T4-binding activity, the affinities for the hormones being of the same order of magnitude. The T4 interaction with these preparations induced difference UV-absorption signals, altered the characteristics of apoA-1 intrinsic fluorescence without affecting the circular dichroism of the protein-hormone system. The binding of spin-labelled T4 to apo-1, apoA-1-HDL or HDI3 caused substantial changes in the shape of the ESR spectrum and an increase in the apparent rotational correlation time. The mobility of the radical fragment of spin-labelled T4 depended on the composition and properties of the protein preparation. The electron spectroscopy data suggest that the T4-HDL interaction occurs via specific mechanisms and that the molecular structures of the complexes formed thereby are not characteristic of other known T4-binding proteins.

[Regulation of the catalytic activity of the monooxygenase enzyme system depending of the substrate structure and phospholipid composition of the model membrane]. / Reguliatsiia kataliticheskoi aktivnosti monooksigenaznoi fermentoi sistemy v zavisimosti ot struktury substrata i fosfolipidnogo sostava model'noi membrany.

A comparative study of pentoxy- and benzyloxyresorufin dealkylation by a monooxygenase enzyme system in dilauroylphosphatidylcholine micelles and in proteoliposomes was carried out. In proteoliposomes whose lipid matrix is formed by double phospholipid mixtures, a cooperative regulation of the oxidation reaction was found. It was shown that the cooperativity of this process depends on the substrate type as well as on the phospholipid composition of the vesicles and decreases during peroxide oxidation of lipids. The results obtained are discussed in terms of protein-protein and protein-phospholipid interactions in the oligomer ensembles consisting of several cytochrome P-450 LM2 molecules.

[The use of hemoglobin for determination of phospholipase A2 activity]. / Primenenie gemoglobina dlia opredeleniia aktivnosti fosfolipazy A2.

A spectrophotometric method is proposed for determining phospholipase A2 activity, which is based on the conversion of hemoglobin into hemichrome under the fatty acid action. The spectral difference between hemoglobin and hemichrome was registered by the difference spectrum with a minimum at 405 nm and a maximum at 423 nm. The absorption value determined as the difference between the spectrum maximum and minimum was proportional to the amount of the fatty acid derived from hydrolysis of phospholipids. The method enables the enzyme activity to be determined directly in the spectrophotometric cell.

Hemoglobin conversion to hemichrome under the influence of fatty acids.

The effect of free fatty acids on the process of hemoglobin conversion and lipid peroxidation has been studied in model systems and erythrocytes. It has been found that methemoglobin and oxyhemoglobin are converted to the low spin oxidized form, namely, reversible hemichrome under the action of fatty acids. In the case of oxyhemoglobin, an increase in the level of active oxygen forms is observed in the system which initiates the formation of primary and secondary lipid peroxidation products. Incubation of erythrocytes with free fatty acids causes the formation of Heinz bodies and is accompanied by an increase of the lipid peroxidation level.

[Determination of blood hemoglobin using sodium dodecyl sulfate]. / Opredelenie gemoglobina krovi s ispol'zovaniem dodetsilsul'fata natriia.

A method for photometric determination of the blood hemoglobin is described. It is based on the transformation of hemoglobin forms into hemichrome ones by sodium dodecyl sulfate, followed by measurement of absorption at 540 nm.

[Initiation of lipid peroxidation as a result of hemoglobin transformation into hemichrome induced by free fatty acids]. / Initsiirovanie perekisnogo okisleniia lipidov v rezul'tate prevrashcheniia gemoglobina v gemikhrom pod deistviem svobodnykh zhirnykh kislot.

The effects of free fatty acids on hemoglobin conversion and lipid peroxidation were studied in hemoglobin-containing liposomes (hemosomes) formed from an equimolar mixture of phosphatidylcholine (PC) and phosphatidylethanolamine (PE). It was shown that in hemosomes oxyhemoglobin is converted into hemichrome by the interaction of saturated fatty acids (arachidic, stearic, palmitic, myristic and lauric). This is accompanied by accumulation of primary and secondary products of lipid peroxidation. All fatty acids, except for lauric acid, have a stabilizing effect on lipid peroxidation in liposomes prepared from an equimolar mixture of PC and PE. The formation of lipid peroxidation products is inhibited by superoxide dismutase, D-alpha-tocopherol, D-mannitol and thiourea. The relationships between hemoglobin conversion and lipid peroxidation in hemosomes under effects of fatty acids were studied. The mechanisms of these reactions are discussed.

[Temperature quenching of cytochrome P-450 fluorescence in proteoliposomes with different physical states of the lipid bilayer]. / Temperaturnoe tushenie fluorestsentsii tsitokhroma P-450 v proteoliposomakh s razlichnym fizicheskim sostoianiem lipidnogo bisloia.

Studies were carried out of temperature quenching of self-fluorescence of cytochrome P-450 in solution and liposomes from natural phosphatidylcholine, dimiristoylphosphatidylcholine, dipalmitoylphosphatidylcholine. The fluorescence spectrum of cytochrome P-450 is a superposition of triptophane and tyrosine components. During protein incorporation into liposomes a significant short-wave shift of the emission spectrum takes place. Temperature dependence of the intensity of cytochrome P-450 self-fluorescence in solution has bends at 30, 45 and 50 degrees C. When protein is incorporated into liposomes the location of bends depends on individual properties of lipids forming the bilayer. Effect of lipid surrounding on temperature conformational rearrangements of cytochrome P-450 molecule is discussed.

[Fatty-acid initiated auto-oxidation of hemoglobin]. / Initsiiruemoe zhirnymi kislotami avtookislenie gemoglobina.

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.

[Interaction of cytochrome P-450 with phospholipids in dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol mixtures]. / Vzaimodeistvie tsitokhroma P450 s fosfolipidami v smesiakh dimiristoilfosfatidilkholina s dimiristoilfosfatidilglitserinom.

ESR and microcalorimetry methods were employed to investigate the thermotropic properties and structure of proteoliposomes that incorporate cytochrome P450 and DMPC-DMPG binary mixtures depending on cytochrome P450 content and phospholipid composition. The microcalorimetry data demonstrated that the incorporation of cytochrome P450 into the phospholipid mixture resulted in bilayer thermal stabilization. The maximum shift of the temperature and proteoliposome transition enthalpy were achieved at the protein/lipid molar ratio of 1:1000 in almost equimolar phospholipid mixture. Using fatty acids that were spin-labeled at different positions (C5, C12, C16), it has been shown that the incorporation of cytochrome P450 into lipid mixtures containing 0-100% DMPG decreases C12 and C16 mobility and increases the C5 order parameter at transition phase (30 degrees C) and liquid crystal phase (37 degrees C) of bilayer. The maximum alteration amplitude of the probes used was not characteristic for the separate DMPC and DMPG but rather for the mixture at the molar ratio close to equimolar value. It is proposed that cytochrome P450 incorporation into the binary mixture initiated the formation of the bilayer crystal-like phase.

[Effector role of fatty acids in the transformation of methemoglobin into hemichrome]. / Effektornaia rol' zhirnykh kislot v protsesse perekhoda metgemoglobina v gemikhrom.

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.