[Thermoinactivation of potato 5-lipoxygenase and effect of phosphatidic acid on activation energy of denaturation].

The investigation aim was to establish the structural-functional relations of individual lipid metabolism components: enzymes--lipoxygenases and membrane phospholipids. Influence of phosphatidic acid (PA)--allosteric activator of potato tuber 5-lipoxygenase (5-LO)--on thermoinactivation thermodynamic parameters of enzyme was studied. It was established that PA changes essentially the 5-LO thermostability, namely activation energy Ea of enzyme denaturation increases several times in this phospholipid presence. Such changes can evidence that PA interaction with 5-LO leads to conformational changes of enzyme probably due to hydrophobic interactions. Obtained results give a possibility to interpret the action mechanism of PA as 5-LO allosteric activator, which can displace the substrate molecules in binding sites and increase the level of formation of specific products of linoleic acid oxygenation by 5-LO.

[Modeling of linoleyl hydroxamic acid influence on lipoxygenases in vitro].

5-Lipoxygenase (5-LO) (1.13.11.12) demonstrates its activity in membrane-associated state. A system in vitro with increasing quantity of mixed micelle of nonionic detergent Lubrol PX and substrate--linoleic acid (LA) was used for understanding of 5-LO catalytic activity mechanism, which depends on the membrane environment. Physical parameters of micelles with molar ratio LA-Lubrol PX = 0.3:1 and micelles with 5-LO inhibitor--linoleyl hydroxamic acid (LHA), LA and Lubrol PX (0.03:0.3:1) were characterized by gel-filtration method on Sephadex G-200. It was determined, that Stock's radii were 4.83-5.79 nm for micelles with total LA--50-2000 microM and average molecular mass--177 000-212 000 Da. The presence of 10 microM LHA has no influence on physical parameters of the system. Influence of LHA on kinetic parameters of LA oxidation reaction catalized by potato tubers 5-LO in characterized mixed micelle system was also studied. Substrate dependences curves of 5-LO LA oxidation steady-state rates under conditions of the mixed micelle with ratio LA-lubrol PX = 0.3:1, LHA-LA-Lubrol PX = 0.03:0.3:1 and LHA-LA-Lubrol PX = 0.12:0.3:1 were typical of the substrate inhibition. The presence of inhibitor had no effect on the number of additional substrate molecules--LA which contact with enzyme-substrate complex and decreased V(max) essentially. To predict further inhibitor transformation in the cell the influence of 13-hydroperoxy- and 13-hydroxy LHA on potato tubers 5-LO and porcine leucocyte 12-LO was investigated. It was established that LHA oxidized forms displayed as no less effective inhibitors of the analyzed enzymes; 13-hydroperoxy LHA efficiency increased by an order (IC50 was 0.7 microM) for 12-LO. The possibility of 5-LO to oxidize inhibitor LHA under 50 microM phosphatidic acid at pH 5.0 was demonstrated.

[Interaction between 5-lipoxygenase and allosteric effector--sodium dodecyl sulfate].

The role of allosteric effector--sodium dodecyl sulfate (SDS) in the lipoxygenase catalysis in micelle system has been studied. The effect of the stable hydrophobic bis-nitroxides, blocking the free radical transformation, on the oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase from potato tuber has been investigated. The inhibiting effect of nitroxide compounds on oxidation of linoleic acid or linoleic alcohol by 5-lipoxygenase depends on SDS concentration. The inhibition percentage is determined by the substrate nature and presence of allosteric effector. The presence of SDS did not lead to an appreciable change in the pKa values of ionogenic enzyme groups. The effect of SDS and micellar system on thermodynamic parameters for thermoinactivation of 5-lipoxygenase was studied. It was found that thermoinactivation rate constants and activation energy of enzyme thermoinactivation were increased in the presence of SDS. It is suggested that interaction of 5-lipoxygenase and allosteric effector--SDS intensifies the dissociation of radical intermediates from the active site of the enzyme. These findings are of physiological significance in the light of the lipoxygenase involvement in the membrane lipid peroxidation.

[Effect of phosphatidic acid on the reaction of linoleic acid oxidation by 5-lipooxygenase from potatoes].

Influence of anionogenic phospholipid of phosphatidic acid (PA) on oxidation of linoleic acid by 5-lipoxygenase (5-LO) from Solanum tuberosum was studied. The influence of PA was studied in micellar system which consisted of mixed micelles of linolenic acid (LK), Lubrol PX and different quantity of enzyme effector PA. The reaction was initiated by addition of 5-LO. It was established that 5-LO had two pHopt. in the presence of 50 microM phosphatidic acid: pH 5.0 and 6.9. In concentration of 50 microM PA was able to activate 5-LO 15 times at pH 5.0. The reaction maximum velocity (Vmax) coincided with Vmax of lipoxygenase reaction without the effector at pH 6.9 under such conditions. It was found that 30-50 microM phospholipid in the reaction mixture decreased the concentration of half saturation by the substrate by 43-67%. The enzyme demonstrated positive cooperation in respect of the substrate, the reaction is described by the Hill equation. Hill coefficient value (h) of the substrate was 3.34 +/- 0.22 (pH 6.9) and 5.61 +/- 0.88 (pH 5.0), that is with the change of pH to acidic region the number of substrate molecules increased and they could interact with the enzyme molecule. In case of substrate insufficiency the enzyme demonstrated positive cooperation of PA, it added from 4 to 3 effectors' molecules at pH 5.0, that is the phospholipid acted as the allosteric regulator of 5-LO. A comparative analysis of the influence of 4-hydroxy-TEMPO displayed, that the level of nonenzymatic processes in the case of physiological pH values was lower by 15-50% in the presence of PA in the range of 30-80 microM than without the effector.

[The effects of alpha-linolenic acid on the functioning of the isolated heart during acute myocardial ischemia/reperfusion].

Many studies indicate that dietary omega-3 polyunsaturated fatty acids (PUFAs) have the cardioprotective properties. But majority of experiments were carried out with using omega-3 PUFAs from marine fish oil. The purpose of this study was to determine effects of the plant-derived omega-3 PUFA (alpha-linolenic acid (a-LA) on postischemic myocardial dysfunction, lipid peroxidation and antioxidant enzymes activity. Male Wistar rats (250-300 g) were divided into 4 groups (n = 10-12 each). In control group (1) were intact rats. The hearts from 2-nd group of animal were exposed to 20 min of global ischemia followed by 40 min reperfusion according to the Langendorff technique. The 3-rd and 4-th groups of animal received of the plant-derived oil (a-LA), which is a precursor of eicosapentaenoic acid and was used as a dietary supplement in dose 0.1 mg/kg per day for 4 weeks. The hearts from 4-th group of animal were also exposed to ischemia/ reperfusion. Analysis of myocardial phospholipid fatty acid content showed that consumption of the plant-derived 6-LA for 4 weeks changes fatty acid profile through incorporation of b-LA in cell membranes. It also reduced content of omega-6 PUFAs in membrane phospholipids. In 3-rd group content of a-LA and EPA were increased by 1.5- and 3.5-times, respectively, whereas content of AA was reduced by 1.7-times. The development of ischemia/ reperfusion in 2-nd group caused increase of free AA content in heart tissue by 3.5-times, whereas in 4-th group this increase was only by 1.4-time. Ischemia/reperfusion of the isolated rat heart in 4-th group was accompanied by reduced leukotriene C4 and thromboxane B2 production in 3-times and 1.9-times, respectively in comparison to 2-nd group. The time of myocardial function recovery after ischemia (heart rate, left ventricular development pressure), was shorter compare to 2-nd group. Also in 4th group end-diastolic pressure and coronary perfusion pressure during reperfusion period were significantly lower. Dietary omega-3 PUFAs resulted in remarkable decrease of reperfusion arrhythmias in 4-th group (in 3.8-times) and limited the oxidative stress through decrease free radical and lipid peroxidation production. In this group of animals the activity of antioxidant enzymes (superoxide dismutase and catalase) after ischemia/reperfusion were higher than in 2-nd group. We suggest that dietary supplement of the plant-derived alpha-LA for 4 weeks have cardioprotective effects similar to the effects of fish oil.

[Inhibiting properties of stable nitroxyl radicals in reactions of linoleic acid and linoleyl alcohol oxidation catalyzed by 5-lipoxygenase].

The inhibiting effects of 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and its 4-substituted derivatives in reactions of linoleyl acid or linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase were investigated. Inhibiting properties of stable nitroxyl radicals in presence of lubrol and SDS were reduced at the transition from TEMPO to 4-hydroxy-TEMPO or 4-amino-TEMPO and increased at use of adamantane-1-carboxylic or 3-methyladamantane-1-carboxylic acid 1-oxyl-2,2,6,6-tetramethylpiperidine-4-yl esters. Enzyme activity at saturating concentrations of inhibitor was not suppressed completely, and decreased up to the certain level determined by the substrate nature. The dependence of partial inhibition efficiency on rotational correlation time of stable nitroxides in model micellar systems were analysed. It was supposed that 5-lipoxygenase inhibition includes the interaction of hydrophobic nitroxide with radical intermediate formed in enzymatic process.

[The effects of omega-3 polyunsaturated fatty acids on lipid peroxidation].

The aim of this study was to evaluate the effects of a diet supplemented with omega-3 polyunsaturated fatty acids (PUFAs) (plant-derived alpha-linolenic acid and eicosapentaenoic and docosahexaenoic acids with alpha-tocopherol (product "Tekom") on the myocardial phospholipid fatty acids composition, lipid peroxidation and activity antioxidant enzymes--superoxide dismutase and catalase in myocardial tissue in control and after ischemia/reperfusion of isolated working rat hearts. Inclusion of omega-3 PUFAs into the animals' diet within 4 weeks demonstrated that alpha-linolenic acid and "Tekom" increased omega-3 polyunsaturated fatty acids content in cardiomyocytes membranes and limited lipid peroxidation (decreased content of congugates of dienes and malone dialdehyde, reduced hemiluminescence of myocardial tissue). Additionally omega-3 PUFAs caused the beneficial effects on activity of antioxidant enzymes in cardiac tissue (increased superoxide dismutase and catalase activity).

[Hydrophobic nitroxyl radicals inhibit linoleyl alcohol oxidation by 5-lipoxygenase]. / Gidrofobnye nitroksil'nye radikaly--ingibitory okisleniia linolevogo spirta 5-lipoksigenazoi.

The linoleyl alcohol oxidation catalyzed by potato tuber 5-lipoxygenase was found to be efficiently inhibited by stable nitroxyl radicals: 1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl 1-bicyclo[2,2,2]octane-1-carboxylate, 1-adamantylacetate, dodecanoate, and octadecanoate. The dependence of apparent IC50 values on the rotational correlation times of times of 4-hydroxy-1-oxyl-2,2,6,6-tetramethylpiperidine and its derivatives in model micellar systems was analyzed. The inhibition mechanism was proposed; it involves the interaction of hydrophobic nitroxyl radical with the intermediate radical enzyme-substrate complex.

[Effect of modification in fatty acid composition of membrane phospholipids on reaction of isolated heart in acute ischemia-reperfusion]. / Vplyv modyfikatsiï zhyrnokyslotnoho skladu fosfolipidiv membran na reaktsiiu izol'ovanoho sertsia pry hostrii ishemiï-reperfuziï.

The aim of this study was to evaluate the effects of a diet supplemented with omega-3 polyunsaturated fatty acids (eicosapentaenoic and docosahexaenoic) and tocopherol, which are the base of a preparation "Tekom", on a composition of myocardial phospholipid fatty acids, as well as on the metabolism of eicosanoids, free radical processes and the contractility of isolated working heart in rats at ischemia/reperfusion. Added to the diet within 4 weeks, "Tekom" induced an increase in the content of omega-3 polyunsaturated fatty acids in membranes of cardiomyocytes, a decrease in vasoactive metabolites of arachidonic acid and limitation of free radical processes. "Tekom" inhibited cardiac arrhythmias in the isolated working hearts of rats and improved the cardiac pump function at ischemia/reperfusion.

[Kinetic and mechanism of interaction of L-cysteine with alkaline phosphatase from calf intestines]. / Kinetika i mekhanizm vzaimodeistviia L-tsisteina so shchelochnoi fosfatazoi iz kishek telenka.

The effect of L-cysteine on activity of hydrophobic forms of calf intestine alkaline phosphatase was investigated. Apparent inhibition constants for mixed type inhibition have been determined. The kinetic results allow supposing that the mechanism of equilibrium establishment between the inhibitor and enzyme involves the initial rapid formation of intermediate complex and a subsequent slower step leading to its stabilization in the substrate binding site. The microscopic rate constants for slow step of interaction of L-cysteine with alkaline phosphatase have been calculated. Effect of pH on apparent inhibition constants and kinetic parameters for enzymatic reaction in the presence of L-cysteine was analysed.

[Characteristics of the aggregative state of the substrate in the reaction of 5-lipoxygenase oxidation of linoleic acid]. / Kharakteristika agregatnogo sostoianiia substrata v reaktsii 5-lipoksigenaznogo okisleniia linolevoikisloty.

5-lipoxygenase (EC 1.13.11.12) oxidizes polyunsaturated fatty acids by molecular oxygen. The enzyme acts in close contact with the cell membranes, which main components are ionic and non-ionic lipids. In order to investigate the kinetic parameters of 5-lipoxygenase reaction in vitro, extremely hydrophobic fatty acid substrate (linoleic acid) should be solubilized in the reaction mixture. We used Lubrol PX ("Sigma" Chem. Co), as a non-ionic detergent consisted of oligoethylene glycol and fatty alcohol. Linoleic acid and Lubrol PX formed mixed micelles thus solubilizing the fatty acid substrate in a buffer with appropriate pH. We have studied the sizes and shapes of mixed micelles Lubrol PX/linoleic acid (aggregates type 1) and Lubrol PX/linoleic acid/SDS (aggregates type 2; SDS was an effective activator of potato tuber 5-lipoxygenase) by means of gel-filtration and laser light scattering techniques. The parameters under investigation were molecular weights, Stocks radii and shapes of the mixed micelles. The average molecular weights and Stocks radii of the mixed micelles type 1 determined by mean of gel-filtration on Sephadex G-200 were 95,142 +/- 5184 Da and 3.45 +/- 0.11 nm, respectively. The same parameters for the mixed micelles type 2 were 73,694 +/- 893 Da and 3.02 +/- 0.02 nm, respectively. The strong similarity in physicochemical parameters for both types of mixed micelles indicated that SDS did not influence the size and shape of mixed micelles of Lubrol PX and linoleic acid. The activatory action of SDS on potato tuber lipoxygenase may be a result of electrostatic effect or direct participation of SDS in enzymatic catalysis. The laser light scattering technique allowed to determine two main fraction of particles in type 1 system with hydrodynamic diameters 2.6 and 5.7 nm and relative contribution to light scattering 13 and 87%, respectively. The particles with d = 5.7 nm were interpreted as the mixed micelles. The particles with d = 2.6 nm were interpreted as isolated molecules of Lubrol PX, linoleic acid and (or) their premicellar aggregates. The data obtained are to be used in creation of reliable physical and mathematical models of 5-lipoxygenase.

[The protective effect of omega-3 polyunsaturated fatty acids on the activity of the isolated rat heart during myocardial ischemia-reperfusion]. / Protektornyi vplyv omeha-3 polinenasychenykh zhyrnykh kyslot na diial'nist' izol'ovanoho sertsia shchura pid chas ishemiï-reperfuziï miokarda.

It was produced plant-derived product, an omega-3 acid-enriched substrate (64%). In our study we tested the influence of this preparation, which is supposed a membrane-modifying agent, on the processes of damage to the isolated heart under conditions of ischemia-reperfusion. Animals took this substrate as nutrient addition to usually everyday diet. We assumed disorders in cardiodynamics and contractile functions of the myocardium (we measured a perfusion pressure in coronary vessel, left ventricular pressure and dp/dt) and in structure of cardiomyocytes. All mentioned parameters was much better after ischemia-reperfusion in hearts from animals which took an omega-3 acid-enriched substrate in course of 4 week before experiments than in hearts from control animals. Conclusions. Omega-3 polyunsaturated acids exert protective effect on functioning and structure of the isolated rat heart during ischemia-reperfusion.

[Inhibitory effect of linoleyl-hydroxamic acid on the oxidation of linoleic acid by 12-lipoxygenase from porcine leukocytes]. / Ingibitornoe vliianie linoleil-gidroksamovoi kisloty na oksilenie linolevoi kilsoty 12-lipoksigenazoi iz leikotsitov svin'i.

Linoleic acid oxidation by 12-lipoxygenase from porcine leukocytes has been studied as affected by linoleyl-hydroxamic acid. Linoleyl-hydroxamic acid has been found to be an effective inhibitor of porcine leucocyte 12-lipoxygenase. Aerobic preincubation of 12-lipoxygenase with 0.1-6 microM of linoleyl-hydroxamic acid led to a time- and dose-dependent inhibition of the enzyme. The inhibitor's concentration able to induce a 50% loss of the enzyme activity with and without 15-min preincubation were 3.5 and 0.65 microM, respectively. Experimental results obeyed a kinetic scheme, which supposed 2 extra substrate molecules bounding with the enzyme-substrate complex in the presence of linoleyl-hydroxamic acid.

[Role of phospholipids in the regulation of activity of porcine leukocyte 12-lipoxygenase]. / Rol' fosfolipidov v reguliatsii aktivnosti 12-lipoksigenazy iz leikotsitov svin'i.

12-Lipoxygenase from porcine leukocytes was partially purified by using of DEAE-Toyopearl chromatography (pH 7.5). Phosphatidylcholine and Phosphatidylinositol in reaction mixtures with mixed micelles Lubrol PX/linoleic acid inhibited the enzyme. The pH-optimum of lipoxygenase reaction in presence of phospholipids shifted into alkaline region. In the absence of phospholipids 3 additional substrate molecules bound with enzyme-substrate complex. In the presence of either phosphatidylcholine of phosphatidylinositol up to 2 substrate molecules bound with enzyme-substrate complex. The phospholipids competed with linoleic acid for one of the enzyme binding centers. A kinetic scheme of 12-lipoxygenase reaction has been proposed: Phosphatidylinositol lowered the values of Ks and Kns of the reaction of linoleic acid oxidation by 12-lipoxygenase, while phosphatidylcholine had opposite effect on these parameters. We suppose that phospholipids can regulate 12-lipoxygenase activity via control of the enzyme affinity to the substrate (polyunsaturated fatty acid).

[Kinetic mechanisms of linoleic acid oxidation by 5-lipoxygenase from Solanum tuberosum L]. / Kinetychni mekhanizmy okyslennia linolevoï kysloty 5-lipoksyhenazoiu iz Solanum tuberosum L.

Linoleic acid oxidation by 5-lipoxygenase from Solanum tuberosum has been studied as affected by sodium dodecylsulfate (Ds-Na). The reaction system consisted of 5-lipoxygenase and mixed micelles of linoleic acid and Lubrol PX. It contained varying amounts of the enzyme effector--Ds-Na. The enzyme showed a pronounced cooperativity, and the reaction was governed by the Hill equation with h = 3.7. On the other side, increasing amounts of Ds-Na added to the system caused a tremendous increase of enzyme activity and simultaneous decline of h, with was proportional to Ds-Na concentration. Ds-Na had dual effect on 5-lipoxygenase--there was an optimal concentration of the compound (0.34 mM Lubrol PX; 0.2 mM LA; 0.13 mM Ds-Na; pH = 6.3) causing the 4-fold highest activation and h = 1.6. The further increase of Ds-Na led to the enzyme inhibition. If Ds-Na was 0.5 mM, h became 1. At this point, each molecule of 5-lipoxygenase bound 3 molecules of Ds-Na and 1 molecule of linoleic acid, thus the total number of occupied binding sites was 4. A kinetic scheme of 5-lipoxygenase reaction has been proposed. It was found that the enzyme's kinetic behaviour could be explaine if assumed an existence of a special noncatalytic binding centre capable of binding several (up to 3) molecules of either substrate, or effector. Such a centre can serve as an anchoring site facilitating the enzyme binding to the surface of lipid aggregates containing insolubilized substrate molecules. Replacing linoleic acid in the binding site, Ds-Na activates the enzyme, possibly due to the much more effective translocation of 5-lipoxygenase to the surface of lipid aggregates. This mechanism can be an universal alternative to the FLAP-type regulation of 5-lipoxygenase activities.

[Effect of 1,25-dihydroxyvitamin D3 on 15-lipoxygenase activity]. / Vliianie 1,25-dioksivitamina D3 na aktivnost' 15-lipoksigenazy.

The influence of 1.25-dihydroxyvitamin D3 on soybean 15-lipoxygenase activity was studied in vitro. 1.25-Dihydroxyvitamin D3 inhibited enzymatic activity in relation to its dose, pH and substrate (linoleic acid) concentration; the findings are indicative of a mixed type of inhibition.

[A hormonally-active form of vitamin D3--1,25-dihydroxyvitamin D3--an inhibitor of leukocyte lipoxygenase]. / Gormonal'no-aktivnaia forma vitamina D3--1,25-dioksivitamin D3-- ingibitor leikotsitarnoi lipoksigenazy.

The effect of the hormonally active vitamin D3 formulation on the activity of porcine leukocyte lipoxygenase was studied. The rate of enzyme inhibition was vitamin dose-dependent and increased if pH values raised, while the vitamin inhibitory effect was decreased as the content of linoleic acid used as a substrate was increased.

Subcellular distribution and properties of rabbit liver aminoacyl-tRNA synthetases under myocardial ischemia.

Subcellular distribution of aminoacyl-tRNA synthetase activities has been studied in normal rabbit liver and under experimental myocardial ischemia (EMI). An increase in the activity of a number of aminoacyl-tRNA synthetases in postmitochondrial and postribosomal supernatants from rabbit liver has been determined 12 hr after EMI. Gel chromatography of the postribosomal supernatant on Sepharose 6B shows that aminoacyl-tRNA synthetase activities are distributed among the fractions with M(r) 1.82 x 10(6), 0.84 x 10(6) (high-M(r) aminoacyl-tRNA synthetase complexes) and 0.12-0.35 x 10(6). In the case of EMI aminoacyl-tRNA synthetase activities are partly redistributed from the 1.82 x 10(6) complex into the 0.84 x 10(6) complex. The catalytic properties of both free and complex leucyl-tRNA synthetases have been compared. KM for all the substrates are the values of the same order in norm and under EMI. A decrease in some aminoacyl-tRNA synthetase activities associated with polyribosomes has been observed 12 hr after EMI. The interaction of aminoacyl-tRNA synthetases with polyribosomes stimulates the catalytic activity of some enzymes and protects them from heat inactivation in vitro. It is assumed that the changes in association of aminoacyl-tRNA synthetases with high-M(r) complexes and compartmentalization of these enzymes on polyribosomes may be related to the alteration of protein biosynthesis under myocardial ischemia.