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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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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作者简介

I. 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

俄罗斯联邦, 54 Odesskaya St., Tyumen, Tyumen region, 625023

А. Abdurakhimov

University of Tyumen

编辑信件的主要联系方式.
Email: az.Abdurakhimov@gmail.com
ORCID iD: 0009-0002-7724-4436

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

俄罗斯联邦, 6 Volodarskogo St., Tyumen, 625003

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

俄罗斯联邦, 186a Sovetskaya St., Almetyevsk, 423450

参考

  1. Andreev V.P., Zachinjaeva A.V. Jendogennye i jekzogennye modifikatory aktivnosti Na,K-ATFazy. Uchenye zapiski petrozavodskogo gosudarstvennogo universiteta. Petrozavodsk: PGU. 2015; 2(147): 7–16. (In Russ.).
  2. Dubrovskij V.N., Abramova N.S. Aktivnost' Na,K-ATFazy razlichnyh otdelov golovnogo mozga krys, podvergnutyh dejstviju immobilizacionnogo stressa i obrabotannyh antiholinjesteraznym preparatom. Vestnik TjumGU. 2017: 65–70. (In Russ.).
  3. Palmgren M.G.1, Axelsen K.B. Evolution of P-type ATPases. Biochimica et biophysica acta (bba) – Vioenergetics. 1998; 1365(1-2): 37–45. doi: 10.1016/S0005-2728(98)00041-3.
  4. Akimova O.A., Kapilevich L.V., Orlov S.N., Lopina O.D. Identifikacija belkov, vzaimodejstvie kotoryh s Na+,K+-ATPazoj aktiviruetsja uabainom. Biohimija. 2016; 81(9): 1269–1279. (In Russ.).
  5. Silkin Y.A., Silkina E.N., Silkin M.Y. The Resistance of Erythrocytes in Some Black Sea Hyposmic Fishes Exposed to the Nonionic Detergents Triton X-100 and Tween-20. Biophysics. 2021; 66(6): 968–973.
  6. Apell H.J., Hitzler T., Schreiber G. Modulation of the Na,K-ATPase by magnesium ions. Biochemistry. 2017; 56(7): 1005–1016; https://doi.org/10.1021/acs.biochem.6b01243.
  7. Isaksen T.J., Hartmann K. Insights into the pathology of the alpha2- Na+/K+-ATPase in neurological disorders; lessons from animal models. Front. Physiol. 2016; 7: 161; https://doi.org/10.3389/fphys.2016.00161.
  8. Tverskoj A.M. Sidorenko S.V., Klimanova E.A. i dr. Vlijanie uabaina na proliferaciju jendotelial'nyh kletok cheloveka korreliruet s izmeneniem aktivnosti Na+,K+-ATPazy i sootnosheniem vnutrikletochnyh koncentracij Na+ i K+. Biohimija. 2016; 81(8): 1112–1121. (In Russ.).
  9. Abramova N.S., Dubrovskij V.N. Aktivnost' transportnyh ATFaz razlichnyh otdelov golovnogo mozga krys, podvergnutyh dejstviju immobilizacionnogo stressa i obrabotannyh antiholinjesteraznym preparatom. Tjumen': vestnik Tjumenskogo gosudarstvennogo universiteta. 2017: 65–70. (In Russ.).
  10. Kravcova V.V., Krivoj I.I. Molekuljarnaja i funkcional'naja geterogennost' Na,K-ATFazy v skeletnoj myshce. 2021; 107(6-7): 695–716. (In Russ.).
  11. Boldyrev A.A. Rol' Na/K-nasosa v vozbudimyh tkanjah (obzor). J. Sib. Fed. Univ. 2008; 3: 206–225. (In Russ.).
  12. Kazennov A.M. Maslova M.N. Osobennosti aktivacii detergentami Na,K-adenozintrifosfatazy golovnogo mozga pozvonochnyh. Zhurnal jevoljucionnoj biohimii i fiziologii. 1980; 16 (50): 430–436. (In Russ.).
  13. Dubrovskii V.N., Orlova L.A. Effect of Detergents on Activity and Magnesium-Dependent Properties of Different Isoforms of Na+,K+-ATPase in the Crude Membrane Fraction of Rat Cerebral Cortex. Bulletin of Experimental Biology and Medicine. 2021; 171(5): 583–587; https://doi.org/10.1007/s10517-021-05279-0.
  14. Chen P.S., Toribara J., Warner T.Y. Microdetermination of phosphorus. Analytical Chemistry. 1956; 28(11): 1756–1758; https://doi.org/10.1021/ac60119a033
  15. Lowry O.H., Rosebrough N.J., Farr A.L. Protein measurement with the Folin phenol reagent. The J. of biological chemistry. 1951; 193(1): 265–275.
  16. Kazennov A.M., Maslova M.N., Shalabodov A.D. Vlijanie otravlenija krys fosfororganicheskim ingibitorom acetilholinjesterazy na aktivnost' Na, K-ATFazy golovnogo mozga. Nejrohimija. 1983. 2(3): 146–152. (In Russ.).
  17. Durfinovа M., Brechtlova M., Liska B., Baroskova Z. K+-p-Nitrophenylphosphatase Activity in Rat Brain and Liver. Physiol. Res. 2009. 58: 121–126; https://doi.org/10.33549/phy-siolres.931191.

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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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