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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1984 Oct;81(20):6521–6525. doi: 10.1073/pnas.81.20.6521

Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+-Ca2+ exchanger) from brain synaptic plasma membranes.

A Barzilai, R Spanier, H Rahamimoff
PMCID: PMC391956  PMID: 6593714

Abstract

A [Na+]-gradient-dependent Ca2+ transporter from brain synaptic plasma membranes has been isolated, purified, and reconstituted into brain phospholipid vesicles. The purification was achieved by sucrose-gradient centrifugation after solubilization of the synaptic membranes in cholate in the presence of a 30-fold excess (by weight) of added brain phospholipids and [Na+]-gradient-dependent Ca2+ loading of the reconstituted vesicles. A 128-fold increase in specific activity of [Na+]-gradient-dependent Ca2+ uptake per mg of protein has been obtained. The purified and reconstituted vesicles took up Ca2+ only in response to an outward-oriented [Na+] gradient. The Ca2+ uptake could be inhibited by dissipation of the [Na+] gradient with nigericin. Successful purification was based on the initial [Na+]-gradient dependency of the Ca2+-transport process, the magnitude of the [Na+]-gradient-dependent uptake, and the presence of purified brain phospholipids. Analysis of the sucrose-gradient-purified reconstituted vesicles on NaDodSO4/polyacrylamide gels showed that the activity coincided with enriched appearance of a 70,000-Da protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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