REVERSAL OF OXIDATIVE-PHOSPHORYLATION IN SUBMITOCHONDRIAL PARTICLES USING GLUCOSE-6-PHOSPHATE AND HEXOKINASE AS AN ATP REGENERATING SYSTEM

Soluble F1-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 half arrow right over half arrow left ATP + glucose (K(eq) = 5 x 10(-4)) During steady-state, the P(i) released in the medium is derived from glucose-6-phosphate which continuously regenerates the ATP hydrolyzed. A membrane potential (DELTA-PSI) can be built up in submitochondrial particles using glucose-6-phosphate 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 (DELTA-G-degrees = -7.0 to -9.0 kcal/mol) and of glucose-6-phosphate (DELTA-G-degrees = -2.5 kcal/mol), the same ratio between P(i) 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 Ca2+ 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 the equilibrium of the reaction towards glucose-6-phosphate formation thus leading to a decrease of the ATP concentration in the medium.