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 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.
D000818:Animals; D001692:Biological Transport; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D010743:Phospholipids / Q000032:analysis; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger; D013574:Synaptosomes / Q000032:analysis* / Q000378:metabolism
D002352:Carrier Proteins; D008565:Membrane Proteins; D010743:Phospholipids; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D002118:Calcium