ADP and Mg 2+ requirement for Ca 2+ accumulation by hog heart mitochondria. Correlation with energy coupling

Abstract

1. 1. The action of ADP and Mg 2+ on the respiration-driven accumulation of calcium phosphate by hog heart mitochondria, in the presence of oligomycin, has been studied. The accumulation is stringently ADP-specific and this requirement does not depend on the substrate used. The site of action of the nucleotide is located at the inner side of the inner mitochondrial membrane. It does not imply the involvement of adenylic translocase. 2. 2. Maximal accumulation of calcium phosphate is observed, even in the absence of exogenous Mg 2+, when ADP is present in the medium from the onset of the incubations. In the absence of ADP, small accumulations of calcium phosphate are obtained, the levels of which do not depend on the addition of Mg 2+. 3. 3. Conversely, once the calcium phosphate accumulation has stopped due to lack of ADP, both this nucleotide and Mg 2+ are needed to reactivate the accumulation process. In this case, the extent of the reactivation is proportional to the Mg 2+ concentration. The fact that it is possible to reverse the inhibition of calcium phosphate accumulation, indicates that this is not due to permanent damage of the mitochondrial membrane. 4. 4. In the initial presence of ADP, calcium accumulation and oxygen uptake appear to be closely correlated. In its absence both parameters are independent: calcium phosphate accumulation stops, whereas oxygen uptake is maximally stimulated. 5. 5. In the absence of ADP, ruthenium red and ethyleneglycol-bis-(aminoethyl)-tetraacetic acid (EGTA) have no effect on the maximally stimulated oxygen uptake. When the nucleotide is initially present, ruthenium red and EGTA promote respiratory control immediately. Once lost, respiratory control in response to ruthenium red is restored when ADP and Mg 2+ are both present. However, with EGTA, Mg 2+ alone is active in re-establishing respiratory control. 6. 6. Exchange between the calcium accumulated and the calcium of the incubation medium has been studied under experimental conditions, which caused any net calcium accumulation to stop. The exchange is inhibited by low concentrations of ruthenium red under conditions where no effect of this inhibitor on the stimulated oxygen uptake can be detected. This contradicts the hypothesis according to which this stimulation could be due to a cyclic movement of Ca 2+. 7. 7. In order to explain the results observed, the lack of symmetry between the respective ADP and Mg 2+ requirements is emphasized. It is postulated that magnesium is stringently required for energy coupling between calcium phosphate accumulation and oxygen uptake. The role of ADP is supposed to be a regulatory one and to consist in an increase of the affinity of the magnesium binding sites for this cation.