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

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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 (M r) of approximately 226,000±20,000sd (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 50mg/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 minimumM r of 224,000 Da. Specific45Ca2+-binding SL proteins withM r 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.

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