Exchange of calcium between muscle Ca²⁺-binding proteins

Sarcoplasmic reticulum isolated from skeletal muscle of the rabbit and the Pacific dogfish (Squalus acanthias) was characterized in terms of its Ca²⁺-uptake at different temperatures. SR from both species showed comparable Ca²⁺-uptake rates at their basal body temperatures (5°C and 37°C, respectively). Only very low percentages of phosphorylase, phosphorylase kinase and phosphatase were found to be associated with the purified vesicles, once these were freed of contaminating glycogen particles. Ca²⁺-fluxes during contraction/relaxation-cycles in muscles were studied in a reconstituted system by measuring the inhibition of the rate of Ca²⁺-uptake by the sarcoplasmic reticulum and of the Ca²⁺-dependent activity of phosphorylase kinase, respectively, by metal-free troponin, troponin-C, parvalbumin and phosphorylase kinase; EGTA was used as an internal standard. Ca²⁺-binding capacities of the various proteins were determined separately by gel filtration experiments. Data obtained agreed with published values for troponin, troponin-C and parvalbumin; for phosphorylase kinase, a value of n- = 8 was found regardless of the state of phosphorylation of the enzyme. Results obtained from both types of experiments established apparent Ca²⁺-affinities in the following decreasing order : phosphorylase kinase ∼ parvalbumin > EGTA > troponintroponin-C, with no difference found between the non-phosphorylated and phosphorylated forms of phosphorylase kinase. Apparent equilibrium constants for these Ca²⁺-complexes, relative to EGTA, were in agreement with published values obtained from classical equilibrium studies. Similar results were obtained with proteins isolated from rabbit and dogfish muscle. No indication of protein-protein interactions affecting Ca²⁺-fluxes could be found in such a reconstituted system, except for the singular case of a slight increased inhibition of phosphorylase kinase by parvalbumin following phosphorylation of the enzyme.