The ultimate objective of this project is the elucidation at the molecular level of those enzymatic phosphate transfer mechanisms in mitochodria which are actively involved in the formation of ATP and other nucleoside triphosphates, and in addition, of those molecular mechanisms which regulate the output of these triphosphates and hence their accessibility to vital biosynthetic processes. To meet this objective phosphate transfer mechanisms in three phases of energy generation in mitochondria of normal or neoplastic tissue are being examined: 1) The phosphorylation of ADP catalyzed by the ATP synthetase, 2) The distribution of high energy phosphate among nucleotides other than adenine by nucleoside diphosphokinase, and 3) The flow of phosphate into mitochondria via a specific carrier. The oligomycin-insensitive ATPase of liver mitochondria, which has been purified in this laboratory, is being examined with respect to its ability to catalyze the following: a) The high affinity binding of ADP and Pi, b) An ADP-ATP exchange, and c) The formation of a kinetically significant phosphorylated intermediate. In addition, the oligomycin- sensitive ATPase of rat liver mitochondria is being examined also for its ability to catalyze the latter reactions. An attempt is being made to elucidate the mechanism by which uncouplers unmask ATPase activity by studying coupled mitochondria from "minimal deviation" hepatomas which are highly deficient in this activity. Studies of the mitochondrial nucleoside diphosphokinase are being continued with emphasis on elucidating its physiological role. Specifically, experiments are being conducted to determine whether multiple forms of this enzyme exist in mitochondria, and whether these forms differ from the cytoplasmic enzyme. In order to better understand the molecular mechanisms which regulate the availability of phosphate to the ATP synthetase, and hence the output of nucleoside triphosphates in general, attempts are being made to purify and characterize the phosphate carrier and demonstrate its role in phosphate transport.