Assay of ATP synthesis using single giant mitochondria

Two assays of the capacity of single giant mitochondria or mitoplasts to phosphorylate ATP from Pi and ADP have been developed. One depends on the placement of a single mitochondrion next to a glycerinated myofibril, by micromanipulation. With the appropriate controls, contraction of the myofibril serves as an indication of ATP synthesis. The other assay similarly requires the isolation of one single mitochondrion but the assay of ATP synthesized uses the luciferin-luciferase reaction with a conventional photometric system. With the latter, we found that either impalement with microelectrodes or electrophoretic microinjection of two dyes, Lucifer Yellow CH or pyranine , into the inner space have no effect on the phosphorylative capacity of mitochondria or mitoplasts. The electric potential across the mitochondrial membrane was monitored during the assay and found to be small and generally positive inside.