Isolation, Purification, and Reconstitution of the Na+Gradient-Dependent Ca2+Transporter (Na+-Ca2+Exchanger) from Brain Synaptic Plasma Membranes

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...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 1984-10, Vol.81 (20), p.6521-6525
Main Authors: Barzilai, Ari, Spanier, Rivka, Rahamimoff, Hannah
Format: Article
Language:English
Subjects:
Animals
Antiporters
Biological Transport
Brain - metabolism
Calcium - metabolism
Carrier Proteins - isolation & purification
Cattle
Cell Membrane - analysis
Cell Membrane - metabolism
Cell membranes
Centrifugation
Detergents
Dialysis
Gels
Lipids
Membrane Proteins - analysis
Molecular Weight
P branes
Phosphates
Phospholipids
Phospholipids - analysis
Rats
Sodium - metabolism
Sodium-Calcium Exchanger
Synaptosomes - analysis
Synaptosomes - metabolism
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Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.81.20.6521