HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Karlish, S. J. D. Articles by Pick, U. Search for Related Content PubMed PubMed Citation Articles by Karlish, S. J. D. Articles by Pick, U.

Biochemistry Department, Weizmann Institute of Science, Rehovoth, Israel

1. (Na,K)ATPase from kidney membranes has been reconstituted into proteoliposomes following solubilization in cholate, by the freeze—thaw sonication procedure described by Kasahara & Hinkle (1977). The method is rapid and convenient.

2. Upon addition of ATP to the exterior medium the reconstituted vesicles sustain high rates of active ²²Na uptake and ⁸⁶Rb efflux with many properties similar to those of the Na/K pump in well characterized cells such as erythrocytes.

3. Observations on both active and passive transport of ²² Na and ⁸⁶Rb indicate that the vesicle population is heterogeneous; about 40 per cent contain Na/K pumps and the remainder seem to be plain lipid vesicles.

4. The major Na⁺- or K⁺-stabilized non-phosphorylated conformational forms of the (Na, K)ATPase, E₁. Na and E₂. (K) respectively, have been investigated in the proteoliposomes, with particular regard to sidedness of the actions of Na⁺ and K⁺.

5. Tryptic digestion of the vesicles reveals the Na⁺- and K⁺-stabilized conformations E₁. Na and E₂. (K) as characterized originally for purified (Na, K)ATPase (Jørgensen, 1975). Controlled trypsinolysis of Tris⁺-loaded vesicles in a Na⁺-or K⁺-containing medium leads to typical biphasic (Na⁺) or simple exponential (K⁺) time courses respectively, for loss of ATP-dependent ²²Na uptake (assayed subsequent to the tryptic digestion in the presence of inophores valinomycin plus FCCP). Tryptic digestion of K⁺- or Rb⁺- or Tris⁺-loaded vesicles suspended in a Na⁺ medium results only in the biphasic (E₁. Na) pattern of loss of ATP-dependent ²²Na uptake.

6. ATP-dependent ²²Na uptake and ⁸⁶Rb efflux are reduced by about the same extent following a short tryptic digestion in a Na⁺-containing medium.

7. Vanadate ions inhibit ATP-dependent ²²Na uptake into the vesicles, at low concentrations (K_0·5 2 x 10^-7 M), following pre-incubation together with Mg²⁺ and K⁺ ions. K⁺ ions in the medium are effective, K⁺ ions within the vesicle are not. Na⁺ ions in the medium prevent inhibition by vanadate+Mg²⁺ but do not reverse inhibition in vesicles pre-incubated with vanadate, Mg²⁺ and K⁺ ions.

8. The results show that the conformational forms E₁. Na and E₂. (K) are stabilized by Na⁺ or K⁺ ions respectively, bound to sites on the Na/K pump normally facing the cytoplasm. The significance of this finding is discussed in relation to the cation transport function of the pump.