Abstract
In conventional in vitro systems, in which the main constituents of reaction media are composed of polyols (mannitol and sucrose) or chloride salt of monovalent cations (KCl and NaCl), the energy conservation system of isolated rat-liver mitochondria responded to added ADP with an extraordinary high sensitivity. Values as low as 10-20μM were regularly obtained as the apparent Km value for ADP, irrespective of whether the mitochondrial response was measured by the activity of oxygen consumption or by the oxidative shift of the oxidation-reduction state of endogenous pyridine nucleotides. The ADP concentration in the liver cell, as estimated by various methods, ranges between 150 and 1,500μ moles per kg wet weight, thus, far exceeding the Km value of mitochondria in the in vitro systems. Two alternatives seem to be possible as the explanation for these facts: the first is that the changes in the ADP concentration is not a physiological determinant in the oxidative phosphorylation activity of mitochondria in situ, and the second is that the Km value for ADP is much higher in mitochondria in situ, thus allowing the ADP concentration still to function as the most important factor in regulation of cell respiration. Direct measurements of the oxidation-reduction state of pyridine nucleotides in the liver in situ by means of in situ organ microfluorometry disclosed a distinctive oxidative response to 10μmoles of dicoumarol administered via the portal vein. Because this uncoupler did not induce the oxidative response in other cell components than mitochondria, it is inferred that the in situ mitochondria, are not in the functional state of “state 3 (as defined by Chance and Williams)”, but in a state between states 3 and 4. This is in keeping with the contention that in situ mitochondria can be reversibly activated by raising the effective concentration of ADP in the cell.
We therefore examined the possibility that the Km value for ADP is much higher in mitochondria in situ than that in mitochondria incubated in conventional systems. This concept was supported by several lines of evidence. In the first place, mitochondria, when combined with the cytosol fraction, exhibited considerably elevated Km values for ADP. Many of the (poly) anionic constituents of the cytosol fraction, such as albumin, ATP and glutathione, mimicked the cytosol effect in that they raised the apparent Km value for ADP. The mode of action of these polyanionic large molecules on the mitochondrial function was analyzed by employing dextran sulfate as an experimental model reagent. The results of such experiments indicate that polyanions which are not permeant to the inner mitochondrial membrane react with the protonated cationic charges on the membrane by the electrostatic force and lead to a competitive inhibition of the activity of adenine nucleotides translocase.
From the foregoing discussions and experimental evidence, it is concluded that (1) ADP can still function as a major regulatory stimulant of the energy conservation reaction in mitochondria in situ,(2)(poly) anionic compounds present in the cytosol component, after an electrostatic interaction with positive charges on the membrane, may competitively inhibit the activity of adenine nucleotides translocase, and this can be one of the mechanisms by which the apparent Km value for ADP is elevated in mitochondria in situ, and (3) thus, the activity of electron transport system can be stimulated either by raising the ADP concentration or by modulating reversible inhibition of the activity of adenine nucleotides translocase.
