Influence of cations and agents on sarcolemmal calcium binding

Calcium binding to fragmented sarcolemma isolated from dog heart was measured by an ultracentrifugation technique. Two classes of binding sites with dissociation constants of 4.2 x 10(-5) M and 1.2 x 10(-3) M were identified. The maximum number of high and low affinity sites were 15 and 452 nmol/mg, respectively. The effects of various cations and drugs on calcium binding were studied in the presence of 0.1 mM total calcium. All cations tested inhibited calcium binding to a certain degree. La3+ (1 mM) and Mn2+ (1 mM) abolished calcium binding to the sarcolemma. The other divalent cation, Mg2+, used at a concentration of 1 mM, produced a partial inhibition Na+ and K+ also depressed calcium binding, with Na+ being the more potent inhibitor. Verapamil produced a depression at concentrations as low as 10(-7) M and abolished calcium binding at 10(-3) M. The effects of these cations and drugs on fragmented sarcolemma appear different from those reported for the isolated sarcoplasmic reticulum. Thus it is unlikely that the calcium binding observed was caused by a contamination of the sarcolemmal preparation by the fragmented sarcoplasmic reticulum. In addition, the calcium binding seems to be on the sarcolemma itself, rather than to the remaining fragments of the basement membrane, because the calcium binding was not significantly modified by pretreatment with purified neuraminidase (0.25 U/g of sarcolemma).