Determination of intramitochondrial pH by means of matrix carbonic anhydrase activity measured with 18O exchange.

The activity of carbonic anhydrase (carbonate dehydratase EC 4.2.1.1)located in the matrix of guinea pig liver mitochondria was measured by monitoring the kinetics of disappearance of "0 from C'sO'80 at chemical equilibrium in bicarbonate buffer as l80 exchanges with "0 in water.This method allows the activity to be measured not only in mitochondria permeabilized by freezing and thawing, but also in situ in intact mitochondria.The activity of the liver mitochondrial enzyme was found to be strongly pH dependent over the range 6.0 to 8.3.The pH dependence was used to monitor intramitochondrial pH in intact mitochondria sus- pended in HC03-/C02 buffer in both energized and deenergized states.In the absence of oxidizable substrate or in the presence of uncoupler, the intramitochondrial pH, pHi, was the same as that of the suspending medium, transmembrane ApH = 0.In the absence of Nethylmaleimide to block movements of endogenous Pi, energization of the mitochondria with succinate gave ApH = -0.2(matrix acidic).In the presence of N-ethylmaleimide, energization with succinate gave ApH = 0.2 (matrix alkaline).During oxidative phosphorylation of ADP, in State 3, ApH = 0.3; in State 4, ApH was -0.2 in the presence of added Pi and 0.1 in its absence.In the presence of exogenous K+ and valinomycin, ApH in energized mitochondria was 0.3; with Na+ replacing K', ApH was -0.2.We conclude that liver mitochondria in HCO3-/Co2 buffer can carry out oxidative phosphorylation and energy-linked cation transport while maintaining transmembrane ApH near zero.It appears that ApH plays a minimal role in energy-linked mitochondrial reactions in the intact organ in vivo which is well supplied with HC03-and COz.' The abbreviations used are: EGTA, ethylene glycol bis(P-aminoethyl ether)-N,N,N',N'-tetraacetate; NEM, N-ethylmaleimide; MS, 225 mM mannitol and 75 m~ sucrose medium; MSB, MS medium containing 25 mM NaHC03; DMO, 5,5-dimethyl-2,4-oxazolylidenedione; std, standard.