Paul V. Blair

Induction of mitochondrial contraction and concomitant inhibition of succinate oxidation by magnesium ions

Abstract The addition of 1 Him MgCl2 to partially swollen rat liver mitochondria respiring in an isoosmotic sucrose-sodium phosphate or sodium acetate medium containing 1 mm EDTA and 15 m m succinate (pH 7.2) initiates contraction, inhibits respiration, and alters the ultrastructural configuration of the inner membrane-cristae-matrix continuum. The maximal extent of contraction (A520 increase) attainable with MgCl2 in the phosphate medium is about 80% of the maximal contraction induced by 2,4-dinitrophenol but exceeds the maximal contraction induced with ADP by about 10%. The extent of mitochondrial contraction and the inhibition of succinate oxidation are dependent on MgCl2 concentration. MgCl2 at 1000 μm immediately inhibits about 70% of the succinate oxidation and initiates maximal extent of contraction concomitant with a distinct configurational change in ultrastructure which appears to differ from that initiated by either ADP or dinitrophenol. MgCl2 at 150 μm does not inhibit the rate of succinate oxidation but it does initiate about 75% of the maximal contraction (A520 increase) attained with 1000 μ m MgCl2. The rate of mitochondrial contraction is dependent on both the phosphate and MgCl2 concentration. CaCl2, in contrast to MgCl2, immediately stimulates succinate oxidation and after a sharp contraction spike of short duration initiates additional expansion of the inner membrane-cristae-matrix continuum. The contraction spike occurs in a reaction system containing either phosphate or acetate. The results are consistent with the notion that Mg2+ and Ca2+ may modulate mitochondrial volume and exert control over certain oxidative processes.

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels.

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/Pi carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.

Correlation of the steady-states of reduction of components of the respiratory chain with phosphorylation states of liver mitochondria.

Summary Rat liver mitochondria were incubated with glutamate plus malate in the presence of 2 mM ATP and inorganic phosphate. Respiration and the steady-state ATP/ADP ratios were altered by addition of controlled amounts of purified ATPase. The redox states of NADH and of cytoehromes b, c-c1, and a-a3 were followed at specified wavelength pairs. NADH, as well as the cytochromes, became more oxidized when electron transfer was stimulated by ATPase. In addition, all of the above components became further oxidized when the phosphorylation state ([ATP/[ADP × Pi]) was further diminished with higher levels of ATPase. The latter occurred without change in the rate of electron transfer. The data are taken to show a thermodynamic interaction between the phosphorylation steady-state and the redox state of the electron transfer carriers. It is emphasized that the state of reduction of respiratory chain carriers measured in extracts from intact tissues cannot be equated with the kinetic steady states defined by Chance and Williams (Adv. Enzymol. 17 , 65–134).

Interdependence between mitochondrial matrix volume and the extramitochondrial ratio of [ATP]/[ADP] [inorganic phosphate].

Abstract ATP or combinations of ATP with EDTA and EGTA can act as chelators to support succinate-driven, phosphate-requiring expansion of mitochondrial inner membrane-matrices. Contraction of these swollen mitochondria can be induced with antimycin, MgCl 2 and ADP. The magnitude of ADP-induced contraction of mitochondria, swollen in the presence of ATP, is dependent on [ADP] and may be altered by the extramitochondrial concentrations of both P i and ATP. In fact, the extent of contraction (+ΔA 520 ) is a linear function of the thermodynamic parameter, −ΔGp (free energy of hydrolysis of ATP), provided excessive concentrations of reactants are not present and the extents of matrix swelling are similar ( e.g. ΔA 520 is about 0.250) before starting contraction with ADP.