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Journal archive > 2009 > N 6 November-December


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) ( 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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