Determination of Intramitochondrial pH and Intramitochondrial-Extramitochondrial pH Gradient of Isolated Heart Mitochondria by the Use of 5,5-Dimethyl-2,4-oxazolidinedione: I. CHANGES DURING RESPIRATION AND ADENOSINE TRIPHOSPHATE-DEPENDENT TRANSPORT OF CA++, MG++, AND ZN++

Abstract

Abstract 1. A newly developed method is described for calculating the intramitochondrial pH of isolated bovine heart mitochondria based on the distribution of 5,5-dimethyl-2,4-oxazolidinedione between mitochondrial compartments, pKa of 5,5-dimethyl-2,4-oxazolidinedione, and pH of the extramitochondrial medium. The measurement of intramitochondrial pH also permitted calculation of the intra- and extramitochondrial pH gradient. 2. Intramitochondrial pH and pH gradient values were calculated for respiration and in ATP-dependent Ca++-, Mg++-, and Zn++-activated bovine heart mitochondria. 3. The intramitochondrial pH of succinate-supported State 4 mitochondria ranged from 7.40 to 7.51 while the extramitochondrial pH ranged from 6.96 to 7.00. The pH gradient ranged from 0.40 to 0.51, depending upon the pH of the extramitochondrial medium. The addition of either 0.565 mm or 6.237 mm Ca++ gave rise to an intramitochondrial pH ranging from 8.31 to 8.58 while the extramitochondrial pH was between 6.70 and 7.00. The pH gradient ranged from 1.51 to 1.75 pH units. Under these conditions the stoichiometry of H+ ejected from and OH- accumulated within the mitochondria was 1.00. 4. When either 20 µm 2,4-dinitrophenol or 1.7 mm phosphate was added to mitochondria activated with 0.565 mm Ca++, the intramitochondrial alkalinization and high pH gradient were neutralized. Acetate (8.3 mm) partially neutralized these effects. 5. No intramitochondrial alkalinization was observed in ATP-supported mitochondria supplemented with 80 nmoles of Ca++ per mg of protein (0.436 mm). 6. The pH gradient of State 3 mitochondria, in the presence of 20 mm Mg++ and permeant anions, was 0.20 pH unit. In the absence of permeant anions, the addition of 10 mm Mg++ to State 4 mitochondria gave rise to a slight intramitochondrial alkalinization. Supplementing succinate-oxidizing (State 4) mitochondria containing 10 mm Mg++ with either 100 or 300 µm Zn++ did not induce any further alkalinization. Ten millimolar Mg++-induced ascorbate oxidation (State 4) gave rise to a slight alkalinization. 7. The pH gradients of State 4 and uncoupled mitochondria were the same. 8. The pH gradients of State 4 mitochondria, in the absence of permeant anions and oxidizing 30 mm succinate, were not different from those oxidizing 5 mm ascorbate in the presence of 100 µm N,N,N',N'-tetramethyl phenylenediamine. 9. The reliability, sensitivity, and limitations of this method for calculating intramitochondrial pH as well as the significance of the findings are discussed.