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Hoffmann, R. A wiki for the life sciences where authorship matters. Nature Genetics (2008)

Partial purification and reconstitution of renal basolateral Na+-Ca2+ exchanger into liposomes.

The Na+-Ca2+ exchange system in renal tubular basolateral membranes was partially purified and incorporated into liposomes. Solubilization of basolateral membranes with 1% cholic acid in the presence of 2.5% soybean phospholipids and proteolytic treatment with Pronase (20 micrograms/ml) as described (Wakabayashi, S., and Goshima, K. (1982) Biochim. Biophys. Acta 693, 125-133) allowed partial purification and reconstitution of the Na+-Ca2+ exchange system into liposomes. The Na+-dependent Ca2+ uptake in the reconstituted liposomes was 25 times higher than the Na+-dependent Ca2+ uptake in the native basolateral membranes. Eadie-Hofstee analysis of the Na+-dependent Ca2+ uptake revealed a Vmax of 201 pmol of Ca2+/mg of protein/45 s and a Km for Ca2+ of 2.7 microM. The stoichiometry (n) of the Na+-Ca2+ exchange system was determined from the Na+ gradient which opposes constant membrane potential so that no net Ca2+ transport occurs. In the presence of constant negative membrane potential, the value for n was 3.09 +/- 0.22, and in the presence of constant positive membrane potential, the value for n was 2.89 +/- 0. 2. Thus, the stoichiometry of the renal Na+-Ca2+ exchange system is approximately 3Na+:1Ca2+.[1]


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