Isolation from Rat Diaphragm of a Calcium-Protein Complex Involved in Excitation-Contraction Coupling

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Abstract
Rat diaphragms depleted of calcium involved in excitation-contraction (E-C calcium) coupling were tagged with 45Ca Seventy percent of the 45Ca was extracted by a 25 mM NaHCO3-50% glycerol solution. Treatment of the muscles with caffeine, shown previously to release E-C calcium, decreased extractable 45Ca to about one-fourth of that of control muscles, indicating that a large fraction of the extracted 45Ca is involved in E-C coupling. On gel filtration, only 10% of the extracted 45Ca was bound to protein. This figure increased to 30% when procaine (which stabilizes E-C calcium) was present in the extraction solution; total extracted 45Ca 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 45Ca at the sites involved in E-C coupling, binds more strongly than 45Ca When 115Cd muscles were extracted with glycerol solution containing procaine, all the extracted ll5Cd was in protein-bound form. One of the extracted proteins formed a precipitin line which was immunochemicaliy identical with rat plasma cardioglobulin-C when tested in gel against an anti-cardioglobulin-C antiserum. This supports our hypothesis that cardioglobulin is the circulating form of a cell membrane calcium transport system.