Flight-muscle mitochondria of Lucilia cuprina and Musca domestica. Estimation of the pyridine nucleotide content and of the response of respiration to adenosine diphosphate

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Abstract
The amounts and reactivity of the pyridine nucleotides of the flight-muscle mitochondria of the flies Musca and Lucilia were estimated by a fluorimetric method and some factors influencing the rates of oxidation of various substrates have been investigated. Particles freshly isolated from both flies contain about 2.5-3.0 [mu]m-moles of total coenzymes/mg of protein, almost all in the oxidized form. About 95% of the total is oxidized diphosphopyridine nucleotide. Incubation with succinate, pyruvate plus malate and (less consistently) a-glycero-phosphate increased the amount of reduced coenzyme. The maximum reduction observed was of 58% of the total, with succinate. Not more than 15% of the pyridine nucleotides were lost from the particles in 15 min. incubation with or without substrate. Adenosine diphosphate increased the respiration of dilute suspensions (about 0.4 mg of protein/ ml) of mitochondria prepared from flies less than 2 weeks old and oxidizing tricarboxylic acid-cycle intermediates or a-glycerophosphate. Stimulations greater than tenfold were obtained. With more concentrated suspensions (1.2-2.0 mg of protein/ml) or older flies, oxidation of [alpha]-glycerophosphate was no longer increased by adenosine diphosphate and was sometimes decreased. qo2 values (with adenosine diphosphate) were much higher for [alpha]-glycerophosphate (200-750 [mu]l. of oxygen/mg of protein/hr.) than for succinate (about 65), pyruvate plus malate (70), malate (3) or glytamate (10). The results emphasize that the respiratory behavior of mitochondrial preparations is influenced greatly by the techniques used for incubation and estimation.