Effect of Polyclonal Antibodies on the Cardiac Sodium‐Calcium Exchanger a

We have previously reported the purification of the cardiac sodium-calcium exchanger from canine myocardium and the generation of polyclonal antibodies against the purified exchanger.' The polyclonal antibodies immunoprecipitated 97% of the sodium-calcium exchange activity from detergent-solubilized sarcolemma and reacted with prominent proteins of 75, 120, and 140 kDa (reducing conditions) on immunoblots of the purified exchanger. Only one major protein, centered at 140 kDa, was detected under nonreducing conditions. Subsequently, antibodies against the 75 , 120-, and 140-kDa proteins were antigen purified and found to immunoprecipitate 92,91, and 83% of the exchange activity, respectively. Furthermore, the antigen-purified antibodies exhibited cross-reactivity with each of the other two prominent proteins. These data are consistent with those reported by Philipson et a L 2 with the exception of minor differences in the molecular weights reported, and suggest that the three proteins are immunologically related and that all are related to the sodium-calcium exchanger. The purpose of the present study was to determine the effect of these antibodies on sodium-calcium exchange activity manifested by sarcolemmal vesicles (70% sealed WO, 0-12% sealed I/O, remainder leaky) from canine ventricle. The vesicles were exposed to increasing concentrations of affinity-purified IgG from preimmune or immune serum. Antibodies from the immune serum stimulated exchange activity 3.5-fold in a dose-dependent manner with half-maximal stimulation at 0.5 pg IgG/pg sarcolemma1 protein (FIG. 1). Conversely, IgG from preimmune serum had little or no effect between 0.01 and 10 pg IgG/pg. A separate experiment was carried out to further test whether the stimulation observed with IgG from the immune serum (anti-NCX) was specific. Four IgG fractions were tested: (1) from the rabbit that was subsequently immunized against the sodium-calcium exchanger (preimmune); (2) anti-NCX; (3) from a rabbit immunized against total sarcolemmal proteins (anti-SL); and (4) from a rabbit immunized against a prominent sarcolemmal protein of 82 kDa enriched by a