Quantitative Aspects of Dog Cardiac Microsomal Calcium Binding and Calcium Uptake

Abstract

Crude microsomes prepared from dog ventricular myocardium by differential centrifugation bound 0.029 µmole of Ca/mg of microsomal protein in the absence of oxalate, and accumulated 1.92 µmole Ca/mg in the presence of oxalate. Purified cardiac microsomes, obtained by sucrose density-gradient ultracentrifugation of the crude microsomes, bound 0.026 µmole Ca/mg and accumulated 2.32 µmole Ca/mg. The total Ca-binding capacity of the myocardium, based on the yields and Ca-binding activities of the crude microsomes, was 0.050 µmole/g wet weight of the ventricle. This value is very nearly equal to the amount of Ca believed to be removed from the actomyosin during relaxation, suggesting that myocardial relaxation may be effected by Ca binding to the sarcoplasmic reticulum. The binding constant for Ca binding to cardiac microsomes is approximately 10⁵ M^-1. Purified cardiac microsomes lowered free Ca from 10^-5 M to approximately 10^-7 M in the presence of oxalate, but Ca uptake proceeded much less rapidly than did Ca binding. Calcium uptake followed first order kinetics. A typical rate constant was 0.24 sec^-1 mg microsomal protein^-1, giving a rate constant of 0.047 sec^-1, for the initial uptake from a solution of 10^-5 M CaCl₂ by 1 g of heart based on the yields of purified microsomes. This rate is significantly lower than that of relaxation in the intact ventricle, and remains so even if the higher yields of crude microsomes are used in the calculation.