Evidence for high molecular weight Na-Ca exchange in cardiac sarcolemmal vesicles.

Abstract

Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na-Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na-Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (Mr) of approximately 226,000 +/- 20,000 SD (n = 6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1:1 with mobile phase buffer containing 50 mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na-Ca exchange activity. Na-Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na-Ca exchange activity were 91 +/- 7 and 68 +/- 11%, respectively. In the peak fraction, Na-Ca exchange specific activity increased two- to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimum Mr of 224,000 Da. Specific 45Ca2+-binding SL proteins with Mr of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose. These data suggest that native cardiac Na-Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na-Ca exchange protein(s) remains incomplete.