Isolation from rat diaphragm of a calcium-protein complex involved in excitation-contraction coupling.

Rat diaphragms depleted of calcium involved in excitation-contraction (E-C calcium) coupling were tagged with Ca. Seventy percent of the Ca was extracted by a 25 HIM NaHCOs-50% glycerol solution. Treatment of the muscles with caffeine, shown previously to release E-C calcium, decreased extractable Ca to about one-fourth of that of control muscles, indicating that a large fraction of the extracted Ca is involved in E-C coupling. On gel nitration, only lOSfc of the extracted Ca was bound to protein. This figure increased to 30% when procaine (which stabilizes E-C calcium) was present in the extraction solution; total extracted Ca remained unchanged. The results suggest that E-C calcium is extracted from the muscle as a calcium-protein complex and is readily dissociated from the protein unless prevented in part by procaine. Cadmium-115, which is shown to replace Ca at the sites involved in E-C coupling, binds more strongly than Ca. When Cd-labeled muscles were extracted with glycerol solution containing procaine, all the extracted Cd was in protein-bound form. One of the extracted proteins formed a precipitin line which was imrnunochemically identical with rat plasma cardioglobulin-C when tested in gel against an anu-cardioglobulin-C antiserum. This supports our hypothesis that cardioglobulin is the circulating form of a cell membrane calcium transport system.