Carlo S. Rossi

Restoration of ATP-induced contraction of pre-treated mitochondria by “contractile protein”

Abstract Ohnishi and Ohnishi (1962 a) have recently isolated from liver mitochondria a protein characterized by an ATPase activity and by physical properties similar to those of the actomyosin of muscle ( Ohnishi & Ohnishi 1962 b ). They suggested that this “contractile protein” constitutes part of a mechano-enzyme system in mitochondria which could be responsible for the swelling-contraction cycle occurring in these particles ( Lehninger, 1962 , Lehninger, 1962 ). It appeared desirable to repeat the experiments of Ohnishi and Ohnishi and to determine if the “contractile protein” possessed a specific activity in the mitochondrial swelling-contraction cycle. Experiments reported in this paper show that the protein fraction extracted from rabbit- or rat liver mitochondria according the procedure of Ohnishi and Ohnishi (1962 a) possesses some but not all of the properties of skeletal muscle actomyosin. However, this protein fraction was found to restore the ATP-induced contraction in mitochondria which had lost that property either after extraction by 0.6 M KCl or after ageing at 0° in isotonic sucrose.

Energy-coupling in mitochondria during resting or state 4 respiration☆

Abstract The resting or State 4 respiration of freshly prepared, intact rat liver mitochondria can support energy-linked accumulation of Ca++ or Sr++ or the oxidative phosphorylation of ADP. However, under these conditions the coupling efficiency is relatively low; about 10% maximal for Ca++, about 20% for Sr++ accumulation, and about 12% maximal for oxidative phosphorylation of ADP.

Active Ion Transport by Mitochondria

The ability of isolated mitochondria to accumulate very large amounts of Ca++ by an active transport mechanism was discovered by V a s i n g t o n and M u r p h y in 1961 [1]. The accumulation process was found to be blocked by respiratory inhibitors and by uncouplers of oxidative phosphorylation, and thus was completely dependent on electron transport and the energy coupling mechanism of oxidative phosphorylation. Significantly, oligomycin does not inhibit Ca++ uptake under these conditions. Later it was found that during the accumulation of Ca++, inorganic phosphate disappears from the medium and also accumulates in mitochondria in amounts that are stoichiometrically related to the Ca++; the molar ratio with which Ca++ and phosphate are accumulated is almost exactly that of Ca++ hydroxyapatite. The amounts of Ca++ and phosphate that accumulate may exceed 200 times the normal mitochondrial content; such accumulation is called “massive loading”. The accumulation of Ca++ and phosphate can also be driven by ATP hydrolysis, and in this case it is not sensitive to respiratory inhibitors, but is inhibited by uncouplers of oxidative phosphorylation and by oligomycin. Ca++ uptake is evidently associated with the dinitrophenolsensitive formation of the first high energy intermediate formed during electron transport, before the point of action of oligomycin.