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ECPB 2015, 71(3): 13–19
Experimental physiology and biochemistry

Time dependence of atp hydrolysis by loach embryonic cells Na+/K+- ATPase under the influence of He-Ne laser


The effects of low-intensity laser radiation are being studied on various biological objects at molecular, cellular and organ levels, however the main mechanism of laser radiation biological action is still not entirely investigated.

Nowadays, the most reasonable hypothesis is that the mechanism of action low-intensity laser radiation can be determined by cytoplasmic membrane structures which absorb photons by acceptor molecules that primarily leads to biochemical reaction activation in the cell. Na+/K+-ATPase is the the plasma membrane marker enzyme selectively inhibited by ouabain, which is the transfer system, that provides the active transmembrane Na+ and K+ ion movement. During enzymatic reactions the protein-enzyme constant dynamic complex reconstruction occurs from one conformational state to another, which can be experimentally detected.

Current approach in the study of enzyme kinetical properties as a part of living cell heterogeneous system, is essential for modern enzymology for the perspective of kinetic modeling, protein enzyme description and prediction under different conditions.

Also, the influence of low-intensity laser irradiation on embryo objects is currently obscure. Previous studies proved that 5 min exposure of low-intensity laser irradiation causes significant increase of MDA contents and activates enzymatic chain of antioxidant protection system of embryo cells at the first hour of development.

Taking all the before-mentioned data into account, the main goal of our research was to investigate the loach embryonic cell membrane Na+/K+-АТPase kinetical properties. Conducting an experiment the embryos (Misgurnus fossilis L.) were exposed by HeNe laser (1,2 mm lens diameter) at a distance of 10 cm from the tube with a wavelength of 632,8 nm and stream density of 1,5 × 102 mW/cm2 for 5 minutes immediately after fertilization and incubated in Holtfreter’s solution.

Na+/K+-АТPase activity was calculated as a difference between the amount of inorganic phosphate released in the presence and in the absence of membrane fragments in the incubation medium, including the loss of endogenous phosphorus Pi in membrane preparation, expressed in micromoles Pi per hour per milligram of protein. The amount of endogenous phosphorus was measured using modified Fiske-Subbarow method, quantitation of total protein content in the membrane preparation was determined by Lowry method.

Studying the kinetics of ATP hydrolysis product accumulation (Pі), embryos were exposed with low-intensity laser radiation for 5 min changing the enzyme reaction duration in the range of 4–20 min (1 min time interval duration).

It has been established that the entire enzyme functional activity arises on 13th–15th minute of enzyme reaction, whereas increase in incubation duration of embryo membranes (from 4 to 20 min) leads to the plateau-like curve formation of enzymatic ATP hydrolysis by incubation time with the maximum of ATPase activity on 14th–16th min of incubation (depending on the stage of embryo division). Inhibitory low-intensity laser radiation effect on Na+/K+-ATPase activity can be implemented through the deviation of ATP enzyme allosteric site binding or changing the main site capability to catalyze the substrate hydrolysis reaction during ATP binding at allosteric site.

Keywords: Na+/K+-АТPase, embryonic cells, kinetics, He-Ne laser

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