Reversal of oxidative phosphorylation in submitochondrial particles using glucose 6-phosphate and hexokinase as an ATP regenerating system

Abstract

Soluble F 1-ATPase and submitochondrial particles are able to bind and to hydrolyze ATP formed from the reversal of the reaction catalyzed by hexokinase: glucose 6-phosphate + ADP = ATP + glucose ( K eq = 5 × 10 −4) During steady-state, the P 1 released in the medium is derived from glucose-6-phosphate which continuously regenerates the ATP hydrolyzed. A membrane potential (Δψ) can be built up in submitochondrial particles using glucose-6-phospate and hexokinase as an ATP-regenerating system. The energy derived from the membrane potential thus formed, can be used to promote the energy-dependent transhydrogenation from NADH to NADP + and the uphill electron transfer from succinate to NAD +. In spite of the large differences in the energies of hydrolysis of ATP (ΔG° = −7.0 to −9.0 kcal/mol) and of glucose-6-phosphate (ΔG° = −2.5 kcal/mol), the same ratio bectween P 1 production and either NADPH or NADH formation were measured regardless of whether millimolar concentrations of ATP or a mixture of ADP, glucose-6-phosphate and hexokinase were used. Rat liver mitochondria were able to accumulate Ca 2+ when incubated in a medium containing hexokinase. ADP and glucose-6-phosphate. The different reaction measured with the use of glucose-6-phosphate and hexokinase were inhibited by glucose concentrations varying from 0.2 to 2 mM. Glucose shifts be equilibrium of the reaction towards glucose-6-phosphate formation thus leading to a decrease of the ATP concentration in the medium.