Solubilization, purification, and reconstitution of the sodium-calcium exchanger from bovine retinal rod outer segments.
We have previously described a method for the solubilization and reconstitution of the cGMP-gated cation channel from the membranes of bovine rod outer segments (Cook, N. J., Zeilinger, C., Koch, K.-W., and Kaupp, U. B. (1986) J. Biol. Chem. 261, 17033-17039). Here we report that not only cGMP but also sodium is capable of releasing entrapped calcium from liposomes reconstituted with total rod outer segment membrane proteins. Other alkali cations tested were unable to induce calcium efflux; therefore, we concluded that the sodium-induced calcium efflux was due to the sodium-calcium exchanger. Sodium was found to activate calcium efflux from these liposomes with an EC50 of approximately equal to 35 mM, comparable to values reported for the sodium-calcium exchanger in native rod outer segment membranes. We found that reconstitution of the sodium-calcium exchanger is quantitative and used this method to assay the exchange protein during purification using conventional protein chromatographic techniques. In this way, we were able to purify and identify as the rod outer segment sodium-calcium exchanger a glycoprotein of apparent Mr = 220,000 to greater than 90% homogeneity. The specific activity of the purified protein at room temperature was 8.2 mumol of Ca2+ exchanged min-1 mg-1 of protein at 50 mM Na+, corresponding to a turnover number of approximately equal to 30 Ca2+ (or 90 Na+) s-1 exchanger-1. The Mr = 220,000 protein reported here appears to be distinct from another protein ("rim protein") with an identical Mr known to exist in these membranes.
Cook
N J
NJ
Universität Osnabrück, Abteilung Biophysik, Federal Republic of Germany.
Kaupp
U B
UB
D000465:Algorithms; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D002850:Chromatography, Gel; D006152:Cyclic GMP / Q000494:pharmacology; D008566:Membranes; D008970:Molecular Weight; D010786:Photoreceptor Cells / Q000032:analysis*; D012374:Rod Cell Outer Segment / Q000032:analysis*; D012964:Sodium / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger; D012995:Solubility
D002352:Carrier Proteins; D019831:Sodium-Calcium Exchanger; D012964:Sodium; D006152:Cyclic GMP