State of translocated Ca2+ by sarcoplasmic reticulum inferred from kinetic analysis of calcium oxalate precipitation.

Uptake of Ca2+ by sarcoplasmic reticulum in the presence of oxalate displays biphasic kinetics. An initial phase of normal uptake is followed by a second phase coincident with precipitation of calcium oxalate inside the vesicles. The precipitation rate induced by actively transported Ca2+ is depressed by increasing the added Ca2+ concentration. This correlates linearly with the reciprocal of precipitation rate. Therefore, a maximal limit rate could be extrapolated at zero Ca2+ (V0). The rate of precipitation, also a function of added amount protein, gives a linear correlation in a double reciprocal plot. Thus, it was possible to estimate the maximal precipitation rate occurring at infinite protein concentration (V infinity). With the combined extrapolated values a maximal expected precipitation rate could be calculated (V infinity 0). Kinetics of calcium oxalate precipitation was studied in the absence of calcium uptake and empirical equations relating the rate of precipitation with the added Ca2+ were established. Entering V infinity 0 in the equations, an internal free Ca2+ concentration of approx. 2.5 mM was estimated. Additionally, it is shown that the ionophore X-537A does not suppress the Ca2+ uptake, if added during the oxalate-dependent phase, albeit the uptake proceeds at a slower rate after the release of approx. 70 nmol Ca2+/mg protein. This amount presumably equals the internal free Ca2+ not sequestered by oxalate, producing a maximal concentration approx. 14 mM. Taking into account low affinity binding of internal binding sites and the transmembrane Ca2+ gradients built up during the uptake of Ca2+, values of free Ca2+ ranging from 3 to 6 mM, approaching those estimated by the precipitation analysis, could be estimated.