Calcium binding properties of beef cardiac troponin.

Abstract

The purpose of this investigation was to compare the Ca2+ binding properties of troponin from cardiac and skeletal muscles since significant differences have been reported in the amino acid sequences of the respective calcium binding subunits. Measurements of Ca”+ binding were made by equilibrium dialysis with metal ion buffers to control the free Ca2+ concentration or by gel filtration in the absence of metal ion buffers. Capacities of 2 and 4 mol of Ca2+ were found/m01 of cardiac and skeletal muscle troponin, respectively. In the presence of MgC12 a single class of binding sites was found for both proteins with an association constant of 3 X lo6 M-‘. In the absence of MgClz, the association constant for cardiac troponin was increased to 4 X lo7 Mm’ with a total capacity of 2 mol of Ca”+. In the absence of MgCl2 a similar increase in the association constant for only two of the binding sites was noted in skeletal muscle troponin, as previously reported (Potter, J. D., and Gergely, J. (1975) J. Biol. Chem. 250, 4628-4633). Assuming competition between Ca2+ and M&+ for the two cation binding sites in cardiac troponin, the binding constant for Mg”+ was 1.6 x lo3 M-‘. A decrease in pH from 7.0 to 6.2 did not alter the capacity or affinity of cardiac or skeletal muscle troponin for Ca2+. Thus, cardiac troponin contains two Ca2+ binding sites which are similar to the two high affinity Ca2+ binding sites of skeletal muscle troponin which also bind M&’ competitively (Ca2+-M$+ sites). Furthermore, a decrease in pH to values which approximate those seen in intracellular acidosis does not lead to a derangement of the Ca2+ binding properties of troponin.