Use of an adenosine triphosphate analog, adenylyl imidodiphosphate, to evaluate adenosine triphosphate-dependent reactions in mitochondria

Abstract

Adenylyl imidodiphosphate (AMP-PNP), an analog of adenosine triphosphate (ATP), was found to be an effective inhibitor of adenine nucleotide translocation in rat liver mitochondria. Inhibition by AMP-PNP was shown to be competitive with ATP. Therefore, studies designed to evaluate the interaction of ATP with mitochondrial adenosine triphosphatase (ATPase) in the presence of AMP-PNP were carried out on submitochondrial particles which lack a membrane barrier between the enzyme and the test medium. The effect of AMP-PNP on the ATP-driven reversed electron transfer reaction in sonically prepared submitochondrial particles was further examined by using oligomycin to induce coupling. The ATPase of oligomycin treated particles did not show significantly different sensitivity to AMP-PNP. Submitochondrial particles which were sensitive to AMP-PNP were less efficient in driving energy-coupled reactions. Results from these studies indicate that uncoupling in mitochondria is not only due to a leaky membrane but may also result from an altered membrane-ATPase association.