A Kinetic Study of Calcium Transport by Heart Mitochondria

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Abstract
Initial velocities of energy‐dependent Ca2+ uptake in cardiac mitochondria were measured with the ethyleneglycol bis(2‐aminoethylether)‐N,N,N′,N′‐tetraacetic acid/ruthenium red quenching technique. Accurate concentrations of free Ca2+ between 1 and 15μM were generated with ATP as a Ca2+ buffer system. Using the method of iterative approximation, the accurate value of the initial velocity of ATP‐driven 45Ca uptake, and the time (t0.5) for half‐maximal uptake can be calculated.The rate of initial Ca2+ uptake increased in response to the rising free Ca2+ concentrations in the medium (in the presence of 0.6–5.0 mM ATP). A maximal rate was obtained between 10 and 15 μM of free Ca2+. For different ATP concentrations (0.6–5.0 mM), the half‐maximal rate of Ca2+ transport (Km) was observed between 4.1 and 7.4 μM Ca2+ (with a mean Km of 5.6 ± 0.6 μ Ca2+; n= 5). Hill plots of the data yield straight lines with an average slope n, of 1.93 ± 0.29, indicating the existence of two binding sites for Ca2+ in the transmembrane transport system. Keeping the free Ca2+ concentration on a constant level (6 μM), the rate of initial Ca2+ uptake also increased continually with rising ATP concentrations (maximum velocity v= 7.1 nmol of Ca2+× mg protein−1× s−1).From energy‐independent calcium‐ion binding studies with cardiac mitochondria, a dissociation constant (Kd) of 26.8 × 10−6M and a total number of external binding sites (n) of 12.6 nmol of Ca2+/mg of protein was obtained.The data are discussed with regard to the physiological role which cardiac mitochondria may play as a reversible storage site during the contraction relaxation cycle of the heart.