It has been found that mitochondria isolated from the flight muscle of the housefly, Musca domestica, are capable of effecting oxidative phosphorylation. A systematic investigation of the factors which regulate this coupling was undertaken. It was found:

1. The molarity of the isolation medium had considerable influence on the morphology of the mitochondria. These physical alterations were associated with changes in oxidation, phosphorylation, and ATPase activity.

2. In addition to an optimum isolation medium, the normal morphology of the mitochondria needed to be further stabilized by serum albumin.

3. A "latent" ATPase activity in insect mitochondria was demonstrated. An inverse relationship was found between oxidative phosphorylation and ATPase activity.

4. Oxygen consumption and the uptake of phosphate were linear with respect to time.

5. A respiratory substrate was necessary for phosphorylation and for maintenance of spatially organized mitochondria.

6. No differences in oxygen uptake were found in the presence or absence of inorganic phosphate.

7. Magnesium was required for optimal oxidative phosphorylation. Calcium and manganese inhibited both respiration and phosphorylation.

8. The addition of cytochrome c had no effect on either oxygen or phosphate uptake.

9. ATP, ADP, or AMP were capable of participating in oxidative phosphorylation, but the glucose-hexokinase trapping system was necessary.

10. Fluoride inhibited the phosphorylation of AMP, but increased P/O when ATP was used. This stimulation was not due to the inhibition of ATPase.

11. Neither arginine nor creatine was phosphorylated.

12. The addition of other isolated fractions of flight muscle to the mitochondrial system had no appreciable effect on respiration or phosphorylation.

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