A Kinetic Study of Calcium Transport by Heart Mitochondria

Initial velocities of energy‐dependent Ca 2+ 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 Ca 2+ between 1 and 15μM were generated with ATP as a Ca 2+ buffer system. Using the method of iterative approximation, the accurate value of the initial velocity of ATP‐driven 45 Ca uptake, and the time ( t 0.5 ) for half‐maximal uptake can be calculated. The rate of initial Ca 2+ uptake increased in response to the rising free Ca 2+ 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 Ca 2+ . For different ATP concentrations (0.6–5.0 mM), the half‐maximal rate of Ca 2+ transport ( K m ) was observed between 4.1 and 7.4 μM Ca 2+ (with a mean K m of 5.6 ± 0.6 μ Ca 2+ ; 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 Ca 2+ in the transmembrane transport system. Keeping the free Ca 2+ concentration on a constant level (6 μM), the rate of initial Ca 2+ uptake also increased continually with rising ATP concentrations (maximum velocity v = 7.1 nmol of Ca 2+ × mg protein −1 × s −1 ). From energy‐independent calcium‐ion binding studies with cardiac mitochondria, a dissociation constant ( K d ) of 26.8 × 10 −6 M and a total number of external binding sites (n) of 12.6 nmol of Ca 2+ /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.