The Na+, K+-ATPase is an ubiquitously expressed P-type of ATPase. Its main role is to maintain Na+ and K+ gradients across the plasma membrane by ATP-driven active transport. The Na+, K+-ATPase was one of the first ion pumps studied because of its importance in maintaining osmotic and ionic balances between intracellular and extracellular environments. Additional extracellular stimuli have been shown to induce internalization of the Na+, K+-ATPase. These include dopamine, parathyroid hormone, hypoxia, hypercapnia and sepsis. The underlying mechanisms appear to involve the activation of several protein kinases, phosphorylation of the N terminus of α-Na+, K+-ATPase and its subsequent ubiquitination.
It has been shown that the activity of Na+, K+-ATPase which plays a key role in the maintenance of intracellular ion homeostasis, osmotic cell balance, transmembrane cell potential, apoptosis regulation, cell proliferation and differentiation is changing under the influence of hormones, growth factors and stress. In addition to the importance of Na+, K+-ATPase in ensuring the function of many tissues (brain, heart and kidney), in the reproductive tract this protein is essential for the embryo development because of its roles in the blastocoel formation and embryo hatching. The study of Na+, K+-ATPase of mammalian embryos mainly performed at the level of gene and protein expression and its biochemical activity has not been investigated. However, the enzyme activity has been indirectly measured by ouabain-sensitive uptake of 86Rb+ of the mouse and cattle embryos. It is also known that the sodium pump collected on the basolateral membranes of mouse morula by using ATP energy, provides the vectorial water transport and blastocoel cavity formation.
Therefore, with a biophysical approach, the research of the Na+, K+-ATPase activity of embryo cells (test system) during embryogenesis with changes in the temperature of the incubation medium in order to determine the activation energy (Ea) of the enzymatic process is relevant. The Na+, K+-АТPase activity has been calculated as a difference between the amount of inorganic phosphate released in the conditions of presence and absence of membrane fragments preparation, expressed in μmoles Pi per minutes per milligram of protein. The amount of endogenous phosphorus has been identified by the modified Fiske-Subbarow method, the quantitation of protein in the membrane preparation has been determined by Lowry method and the activation energy – in the Arrhenius coordinates. The activity of ouabain-sensitive ATPase under the change in the incubation medium temperature gradually increased at the stages of embryos development by an average of 23.4 ± 1.6% as compared with the control group. The maximum value of the enzymatic activity of Na+, K+- pump of the embryos has been defined at the 8-stage of blastomers division (270 min). At the 6th hour of the development, no significant changes in the activity of Na+, K+-ATPase has been observed in comparison with the previous stage of the development.
The results have shown that with the temperature change (10oC) of the incubation medium, the Na+, K+-ATPase activity increased significantly at the investigated stages of the development. For Na+, K+-ATPase, a nonlinear dependence of the enzymatic activity on the temperature in the Arrhenius coordinates was found; the activation energy is 10.6 ÷ 20.8 kJ / mol. The calculated values of the Na+, K+-ATPase activation energy of the loach embryos during embryogenesis are consistent with the existing data and are likely due to the binding strength of the investigated ATP-hydrolase to membrane lipids during the embryonal cell division.
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