Removal of "tightly bound" nucleotides from phosphorylating submitochondrial particles

Phosphorylating submitochondrial particles from beef heart (ETPH) prepared here contained about 2.4 nmol of ATP and 1.9 nmol of ADP/mg of protein after repeated washing of the particles. Essentially all of the "tightly bound " ATP and ADP was removed by trypsin treatment. The trypsin-treated ETPH had increased ATPase activity, undiminished NADH oxidase and succinate oxidase activity, but energy-coupling activity (ATP-driven reversed electron transfer) was abolished. Removal of half the ATP and ADP occurred at low levels of trypsin and was associated with loss of half of the coupling activity. Gel filtration of ETPH in high ionic strength buffer also removed ADP and ATP from the particles, resulting in loss of energy-coupling activity, while ATPase activity was increased. The results support the contention that the tightly bound ADP is essential in energy coupling in mitochondria. Tightly bound ATP may also play an essential role.