Kinetics of mitochondrial calcium transport. I. Characteristics of the sodium-independent calcium efflux mechanism of liver mitochondria.

The kinetics of sodium-independent calcium efflux from liver mitochondria has been studied over the range of calcium loads from 2 to 60 nmol/mg with emphasis on the lower portion of this range. A procedure has been developed through which mitochondria may be depleted of endogenous calcium (initially in the range of 6-10 nmol/mg following preparation) to values as low as 2 nmol/mg, without involving substrate depletion or de-energization. Mitochondria depleted of calcium by this technique are more resistant to the calcium-induced permeability transition than are those depleted by the older procedures and are therefore appropriate for the kinetics studies. Calcium depletion is necessary in studying the kinetics of sodium-independent calcium efflux in order to bring efflux to a rate considerably less than 50% of the saturation rate. The results of these studies show cooperativity with a Hill coefficient of 1.9 +/- 0.2. They have been fit to an equation representative either of a nonessential activation mechanism with a single transport site or of an Adair-Pauling mechanism with two transport sites. From the fit of the data to this equation, a Vmax of 1.2 +/- 0.1 nmol/mg/min and a concentration of half-maximal activity of 8.4 +/- 0.6 nmol/mg have been obtained. The possible role of phosphate in controlling the Vmax of this transporter has been evaluated by measuring efflux as a function of calcium load at three different concentrations of total inorganic phosphate: 20 microM, 120 microM, and 1 mM. Failure of the maximum transport velocity to decrease with increasing inorganic phosphate indicates that the extreme flatness of the saturation portion of the velocity versus calcium concentration curve observed is not the result of precipitation of calcium with inorganic phosphate but is an inherent property of this efflux mechanism.