The effect of sodium deoxycholate and Twееn-20 on the activity and properties of NA+/K+-ATPase in the mixed membrane fraction of the cerebral cortex of rats

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Abstract

Interoduction. The present work extends the understanding of the activity of ouabain-sensitive (α2 and α3) and ouabain-resistant (α1) Na+/K+-ATPase isoforms in the mixed membrane fraction of rat cerebral cortex in the presence of various detergents.

The aim of the study: to determine the activity of ouabain-sensitive (α2 and α3) and ouabain-resistant (α1) Na+/K+-ATPase isoforms by changing the concentration of Mg+2 ions in the incubation medium and using the detergents sodium deoxycholate and Tween-20.

Material and methods. To determine Na+/K+-ATPase activity, we used the method of A. Kazennova et al. The reaction was triggered by adding homogenate of cerebral cortex incubated with detergents; the samples were incubated for 30 minutes in a water bath at 370 C. The activity of ouabain-sensitive isoforms was determined by adding 200 μmol of ouabain to the incubation mixture. The activity of ouabain-sensitive isoforms was determined by adding 200 μmol ouabain to the incubation mixture, and the activity of ouabain-resistant isoforms was determined as the difference between the total Na+/K+-ATPase activity and the activity of ouabain-sensitive isoforms. ATPase activity was determined by inorganic phosphate (Pi) increment as the difference between total ATPase activity, magnesium ATPase activity, in reaction with ammonium molybdate and tin chloride in the presence of ascorbic acid as a reducing agent.

Results. At optimal concentrations of detergents Dox-Na (1.25 mg/mL) and Tween-20 (6.5 mg/mL), both ouabain-sensitive and ouabain-resistant Na+/K+-ATPase isoforms are characterized by normal activity dynamics from the concentration of Mg2+ ions. Consequently, the use of detergents at given concentrations does not change the ability of enzymes to conformational rearrangements during the reaction cycle.

It was found that both used detergents allow to reveal a significant percentage of latent activity of ouabain-sensitive (α2 and α3) and ouabain-resistant (α1) Na+/K+-ATPase isoforms in the mixed mebran fraction of the cerebral cortex of rats.

Conclusion. The study of Mg2+-dependent properties revealed a similar character of the curves of dependence of enzyme activity on the concentration of Mg2+ in the incubation medium for all Na+/K+-ATPase isoforms studied

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About the authors

I. R. Aitmukhambetova

Tyumen State Medical University Ministry of Health of the Russian Federation

Email: ilnara.serikova.01@bk.ru
ORCID iD: 0009-0001-8894-5871

Assistant of Department of Biological Chemistry, Institute of Pharmacy

Russian Federation, 54 Odesskaya St., Tyumen, Tyumen region, 625023

А. А. Abdurakhimov

University of Tyumen

Author for correspondence.
Email: az.Abdurakhimov@gmail.com
ORCID iD: 0009-0002-7724-4436

Student, Institute of Agricultural and Environmental Biology (X-BIO)

Russian Federation, 6 Volodarskogo St., Tyumen, 625003

А. А. Pokhaznikovа

Almetyevsk State Technological University "Higher School of Oil"

Email: sandra190009@gmail.com
ORCID iD: 0000-0001-7450-8876

Engineer of The Advanced Engineering School

Russian Federation, 186a Sovetskaya St., Almetyevsk, 423450

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2. Fig. 1. Activity of α1-isoform of Na+/K+-ATPase in the mixed membrane fraction of rat cerebral cortex preincubated with Dox-Na and Tween-20 detergents at different concentrations. Here and further in the pictures: *, **, *** – differences with control are statistically reliable at p < 0.05, p < 0.01 and p < 0.001, respectively, +,++,+++ – differences are reliable with the previous value at p < 0.05, p < 0.01 and p < 0.001

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3. Fig. 2. Activity of α3-isoform of Na+/K+-ATPase in the mixed membrane fraction of rat cerebral cortex preincubated with Dox-Na and Tween-20 detergents at different concentrations

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4. Fig. 3. Total activity (α2 and α3-isoform) of Na+/K+-ATPase in the mixed membrane fraction of rat cerebral hemispheric cortex preincubated with Dox-Na and Tween-20 detergents at different concentrations

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5. Fig. 4. Na+/K+-ATPase activity at different magnesium concentrations in the incubation medium at peak detergent concentrations (Dox-Na, 1.25 mg/mL; Tween-20, 6.5 mg/mL): A – activity of ouabain-resistant isoform (α1) of Na+/K+-ATPase; B – activity of ouabain-sensitive isoforms (α2 and α3) of Na+/K+-ATPase

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