The effect of temperature and chronic ethanol feeding on the proton electrochemical potential and phosphate potential in rat liver mitochondria

Abstract

The relationship between the proton electrochemical potential ( Δ \\ ̃ gm H ) and the maximal free energy of ATP hydrolysis ( ΔG P) in coupled respiring rat liver mitochondria was investigated as a function of temperature and chronic ethanol-feeding. The flow dialysis method was utilized to measure the temperature dependence of Δ \\ ̃ gm H from the uptake of 86Rb (in the presence of valinomycin) and [ 14C]DMO. ΔG P in state 4 was determined by a null-point titration of the reversible, H +-coupled ATPase against the phosphate potential. Δ \\ ̃ gm H increases with temperature from 196 mV at 10°C, to 217 mV at 40°C. The maximal ΔG P at state 4 decreases as a function of temperature from 67.8 kJ/mol at 10°C, to 54.8 kJ / mol at 40°C. As a result, the ratio ΔG P Δ \\ ̃ gm H decreases with temperature from 3.56 at 10°C to 2.60 at 40°C. Similar studies with mitochondria from rats which were chronically fed with ethanol show that, while ΔG P at state 4 decreases in these rats from 61.2 to 56.0 (25°C), the Δ \\ ̃ gm H is essentially unchanged at 212 mV. Thus the ratio ΔG P Δ \\ ̃ gm H in ethanol-fed rats at 25°C is 2.77 as compared with 2.97 in control. Similar reduction of ΔG P was observed in inverted inner membranes from ethanol-fed rats. Both the temperature dependence of ΔG P Δ \\ ̃ gm H and the effect of ethanol-feeding cannot be easily explained by the chemiosmotic hypothesis which postulates that Δ \\ ̃ gm H is the only driving force for ATP synthesis. In contrast, a parallel coupling model, which postulates that intramembrane proton transfer from redox pumps to ATPase is mediated by the formation of dynamic aggregates of the mitochondrial innermembrane proteins, can easily accommodate these findings. Accordingly, the temperature effect is due to weakening of these fragile aggregates, while the ethanol-feeding effect is the result of reduced concentration of active pumps, which decrease the frequency of formation of functional aggregates.