The energy-dependent accumulation of phosphate by blowfly mitochondria and its effect on the rate of pyruvate oxidation

During an investigation of the kinetic properties of the NAD-linked isocitrate dehydrogenase (NAD-IDH) of mitochondria derived from the thorax of Caliphora vomitora (a species of blowfly) it became apparent that this enzyme was inhibited by ATP and NADH 2 and was activated by ADP, Mg 2+, isocitrate and phosphate. A particularly striking feature was the dependence on phosphate. A plot of activity versus concentration resulted in a sigmoidal curve. At 5mM phosphate the enzyme produced 4–5% of its maximal activity and at 30mM 80%. Sacktor & Hurlbut (1966) have reported that at rest the phosphate content of whole blowfly thorax is 6.8μmoles/gm. wet wt. and in activity 7.5μmoles/gm. In this paper it is shown that isolated blowfly mitochondria are able to accumulate phosphate in a reversible energy-dependent fashion. The concentration in the intramitochondrial water can be as high as 4 times that in the suspending medium. The significance of this finding is illustrated in terms of the kinetic and steady-state response of O 2 uptake and respiratory carriers of intact mitochondria to ADP, phosphate and uncoupling agents. The conclusion is drawn that NAD-IDH activity is the prime controlling factor in the rate of O 2 uptake during pyruvate oxidation by blowfly flight-muscle mitochondria.