Sodium-sensitive calcium binding to sarcolemma-enriched preparations from canine ventricle.
Calcium binding to sarcolemma-enriched preparations from canine ventricle was evaluated. The preparation was exposed to calcium and 45Ca at physiological ionic strength, pH 7.4, for 15-18 hours at 5 degrees C. Bound calcium was separated from free by filtration and washing of the filter with solutions containing calcium and LaCl3. After equilibration at 5 degrees C, exposure to 37 degrees C caused an irreversible loss of binding. Monovalent cations (157 mM) reduced calcium binding: Na+ much greater than Li+ greater than Cs+ greater than K+ greater than Rb+ approximately equal to choline. In 1 microM calcium, divalent cations (3 mM) reduced binding: Sr++ greater than Ba++ greater than Mg++ approximately equal to Mn++. At 1-300 microM calcium, inhibition of the sodium-sensitive component of binding was characterized by I50's of 3.2-9.5 mM sodium. Comparison of binding by centrifugation versus filtration suggested that the sodium-sensitive component resided on constituents within the membrane vesicles. Calcium binding in 1 mM ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid at pH 7.1 and 5 degrees C, revealed a single species of sodium-sensitive calcium-binding sites: Kd = 0.052 microM and Bmax = 6.73 nmol/mg. In 3 mM magnesium, the Kd was 0.205 microM and the Bmax was 9.03 nmol/mg. Nearly complete inhibition of binding was observed as sodium was raised from 1 to 10 mM. Thus, a substantial number of calcium-binding sites were detected at 5 degrees C in 3 mM magnesium at physiological ionic strength and pH 7.1. The affinity of these sites was in the range necessary to modulate intracellular free calcium. The sensitivity to sodium was at the lower end of the range estimated for intracellular sodium.
D000818:Animals; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002498:Centrifugation; D004285:Dogs; D004533:Egtazic Acid / Q000494:pharmacology; D005374:Filtration; D006352:Heart Ventricles / Q000166:cytology; D009206:Myocardium / Q000166:cytology*; D012508:Sarcolemma / Q000378:metabolism*; D012964:Sodium / Q000502:physiology*
D000001:Calcimycin; D004533:Egtazic Acid; D012964:Sodium; D002118:Calcium