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Seasonal Changes in Microsomal Fraction Enriched with Na,K-ATPase from Kidneys of the Ground Squirrel Spermophilus undulatus

E. V. Basevich, O. D. Lopina, and A. M. Rubtsov*

Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, 119991 Moscow, Russia; fax: (495) 939-3955; E-mail: am_rubtsov@mail.ru; basevichev@mail.ru

* To whom correspondence should be addressed.

Received June 24, 2010; Revision received August 5, 2010
The Na,K-ATPase activity in microsomal fraction isolated from kidneys of winter hibernating ground squirrels was found to be 1.8-2.0-fold lower than that in active animals in summer. This is partially connected with a decrease in Na,K-ATPase protein content in these preparations (by 25%). Using antibodies to different isoforms of Na,K-ATPase α-subunit and analysis of enzyme inhibition by ouabain, it was found that the decrease in Na,K-ATPase activity during hibernation is not connected with change in isoenzyme composition. Seasonal changes of Na,K-ATPase α-subunit phosphorylation level by endogenous protein kinases were not found. Proteins which could be potential regulators of Na,K-ATPase activity were not found among phosphorylated proteins of the microsomes. Analysis of the composition and properties of the lipid phase of microsomes showed that the total level of unsaturation of fatty acids and the lipid/protein ratio are not changed significantly during hibernation, whereas the cholesterol content in preparations from kidneys of hibernating ground squirrels is approximately twice higher than that in preparations from kidneys of active animals. However, using spin and fluorescent probes it was shown that this difference in cholesterol content does not affect the integral membrane microviscosity of microsomes. Using the cross-linking agent cupric phenanthroline, it was shown that Na,K-ATPase in membranes of microsomes from kidneys of hibernating ground squirrels is present in more aggregated state in comparison with membranes of microsomes from kidneys of active animals. We suggest that the decrease in Na,K-ATPase activity in kidneys of ground squirrels during hibernation is mainly connected with the aggregation of proteins in plasma membrane.
KEY WORDS: hibernation, Na,K-ATPase, membrane microviscosity, protein aggregation, kidney, ground squirrel Spermophilus undulatus

DOI: 10.1134/S0006297910110143