THE SOLUBILIZATION OF MYOFIBRILLAR PROTEINS BY CALCIUM-IONS

The effect of elevated levels (30 mM) of Ca2+ and other divalent metal ions on rabbit psoas myofibrils was studied to determine whether these caused solubilization of structural proteins and if so whether the effect was due to salting-in or to proteolytic fragmentation resulting from activation of calpains. Incubation of myofibrils in 30 mM CaCl2 at either pH 5.6 or 7.0 did not cause any apparent solubilization of the major Z-disc proteins, but there was an immediate (< 1 min) solubilization of C-protein and troponin I together with small amounts of Mr 80 000 protein, troponin T and tropomyosin. Longer incubations with CaCl2 extracted little additional C-protein but there was a steady increase with time in the solubilization of proteins with Mr values of 45 000 and 42 000, troponin T, tropomyosin and troponin I. Another high molecular weight protein of Mr 3-400 000 was extracted at pH 7.0 but not at pH 5.6. Similar results were obtained on incubation with 30 mM MgCl2. In contrast to these findings, the same concentration of ZnCl2 caused no detectable solubilization of myofibrillar proteins. The inclusion of proteinase inhibitors, E64, leupeptin, pepstatin or PMSF did not prevent the immediate solubilization of proteins. This showed that the solubilization of the proteins by Ca2+ ions was due to salting-in rather than to proteolytic action by calpains.