Steady state kinetics of energy-dependent Ca2+ uptake in rat liver mitochondria.

The divalent cation ionophore A23187 has been used to examine the steady state kinetics of energy-dependent Ca2+ uptake with /3-hydroxybutyrate as oxidizable substrate, and these results have been compared to the kinetic parameters obtained with succinate as the available energy source. The kinetics are sigmoidal with the free Ca*+ concentration at half-maximal respiratory stimulation (K,,,,) equal to 1.4 FM c/3-hydroxybutyrate) versus 3.1 PM (succinate). The respiratory V,,,, is limited by the maximal rate of substrate oxidation which is influenced by the ionic composition of the medium. With /Ll-hydroxybutyrate, Mg2+ is a potent allosteric inhibitor, increasing the K,,.j for Ca”+ to 17.8 PM (25”) at 2.0 mu added MgCl,. Magnesium inhibition is approximately 2-fold greater at 35”. When succinate is the oxidizable substrate, the inhibitory effects of Mg’+ are similar in magnitude, but the measured values reflect the differences in K,,5 for Ca2+ seen in the absence of added cations. The respiratory V,,, is a function of the incubation temperature, but the K,,.j for Ca2+ is affected only when succinate is the energy source. With P-hydroxybutyrate above 20”, Arrhenius plots of the respiratory VmaX or uncoupled respiration were linear with an activation energy of 18.7 kcallmol. The same Arrhenius plots with succinate were not linear, giving activation energies of 8.6 and 13.1 kcabmol above and below 23”, respectively. In contrast, ADP-stimulated respiration resulted in linear Arrhenius plots regardless of oxidizable substrate. The V,,, (succinate) is enhanced 3-fold over the pH range 6.2 to 7.8 and the K,,,, for Ca2+ progressively increased from 0.5 (pH 6.6) to 3.0 PM (pH 8.2) at 35”. With P-hydroxybutyrate, the V,,,,, was much less affected by pH (40%) and the K,.j did not change. Increasing concentrations of the phosphate transport inhibitor, N-ethylmaleimide, progressively inhibited the succinate-supported respiratory V,,,. Inhibition was partial, specific for N-ethylmaleimide, and the K,,, for Ca”+ was not affected. The results suggest that N-ethylmaleimide is acting at a site near the Ca2+ transport system. Thus, the steady state kinetic data reflect the properties of the Ca2+ transport system plus the available energy source and possibly the number of energy-conserving sites involved in Ca2+ transport. The role of Mg2+ as a physiological regu-