Permeability Properties of the Inner Membrane of Mung Bean Mitochondria and Changes during Energization

Abstract

The permeability properties of the inner membrane of mung bean mitochondria were studied by osmotic swelling techniques. Rapid mitochondrial swelling was observed in isotonic ammonium phosphate, which indicated that an active phosphate/hydroxyl antiporter was present. The phosphate carrier was specifically inhibited by sulfhydryl reagents. Mitochondria did not swell in isotonic ammonium salts of malate, succinate, or fumarate, either in the presence or absence of 10 millimolar phosphate. Additionally, no swelling was observed in ammonium citrate upon addition of malate plus phosphate. Consequently, no evidence was obtained with the osmotic swelling technique for a coupled exchange of phosphate for dicarboxylic acids across the membrane.On the basis of valinomycin-induced swelling in potassium salts, suggestions were obtained that chloride, sulfate, and phosphate anions permeated more rapidly than acetate anions. Sulfhydryl reagents increased the rate of valinomycin-induced swelling in potassium phosphate, but had no effect on swelling in potassium acetate.Tributyltin induced a low rate of mitochondrial swelling in KCl in the absence of substrates, which indicated the presence of a low activity (Na(+)) K(+)/H(+) antiporter in the membrane. In the presence of NADH, rapid swelling, followed by a contraction, was observed upon addition of tributyltin. Swelling was insensitive to uncouplers, whereas contraction was uncoupler-sensitive. O(2) uptake (state 4) was greatest (3- to 4-fold stimulation) during the contraction phase, which indicated that the observed contraction was coupled to the pH gradient formed during electron transport. The results suggested that energization increased the activity of the (Na(+)) K(+)/H(+) antiporter in the inner mitochondrial membrane.