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Journal archive > 2009 > N 6 November-December

MODELING OF LINOLEYL HYDROXAMIC ACID INFLUENCE ON LIPOXYGENASES IN VITRO

T. D. Skaterna, V. M. Kopich, V. M. Tsernuk, O. V. Kharchenko

Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Sciences of Ukraine, Kyiv;
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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 µM and average molecular mass – 177 000–212 000 Da. The presen­ce of 10 µM LHA has no influenced 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 Vmax 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 (ІС50 was 0.7 µM) for 12-LO.
The possibility of 5-LO to oxidize inhibitor LHA under 50 µM phosphatidic acid at pH 5.0 was demonstrated.

Key words: 5-lipoxygenase, 12-lipoxygena­se, linoleic acid, linoleyl hydroxamic acid, inhibitor, phosphatidic acid, micelles.

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© The Ukrainian Biochemical Journal