Revealing of Proteins Interacting with Na,K-ATPase

O. A. Akimova, N. V. Dolgova, N. V. Mast, A. M. Rubtsov, and O. D. Lopina^*

Department of Biochemistry, Faculty of Biology, Lomonosov Moscow State University, Moscow 119992, Russia; fax: (095) 939-3955; E-mail: od_lopina@mail.ru

^* To whom correspondence should be addressed.

Received December 11, 2002; Revision received March 12, 2003

---

Proteins interacting with alpha1beta1-type of Na,K-ATPase were revealed in pig kidney outer medulla and duck salt glands using three different methods (immunoprecipitation, protein overlay, and chemical cross-linking). Immunoprecipitation was performed after solubilization of protein homogenate with Triton X-100 so that both membrane and cytosol proteins bound to Na,K-ATPase could be revealed. Two other methods were used to study the interaction of cytosol proteins with purified Na,K-ATPase. The sets of proteins revealed by each method in outer medulla of pig kidney were different. Proteins interacting with Na,K-ATPase that have molecular masses 10, 15, 70, 75, 105, 120, and 190 kD were found using the immunoprecipitation method. The chemical cross-linking method revealed proteins with molecular masses 25, 35, 40, 58, 68-70, and 86-88 kD. The protein overlay method revealed in the same tissue proteins with molecular masses 38, 42, 43, 60, 62, 66, 70, and 94 kD.

---

KEY WORDS: Na,K-ATPase, protein-protein interactions, immunoprecipitation, chemical cross-linking, protein overlay

---