J. Biol. Chem., Vol. 260, Issue 29, 15473-15476, 12, 1985
Partial purification and reconstitution of renal basolateral Na+-Ca2+ exchanger into liposomes
Z Talor and JA Arruda
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+.
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