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

¹ Laboratory of Kidney and Electrolyte Metabolism, National Heart and Lung Institute and the Biology Branch, National Cancer Institute, Bethesda, Maryland 20014

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.

Submitted on May 20, 1971
Accepted on August 5, 1971