The pathways and fluxes for phosphoryl transfer to and from the guanine nucleotide pools within mitochondrial matrix is being determined by tracer experiments and kinetic modeling. Mitochondria will be isolated from tissues and species which differ in the presence and absence of phosphoenolpyruvate carboxykinase and in the use of ATP/ADP vs. GTP/GDP by succinate thiokinase. Matrix preparations from mitochondria are assayed for nucleoside diphosphate kinase, nucleoside monophosphate kinases including adenylate kinase, GTP-AMP phosphotransferase, and guanylate kinase, and GTP-utilizing enzymes such as those which activate medium-chain fatty acids. In these assays, HPLC is used to separate and quantitate all nucleotide substrates and products. The results will be used to develop a kinetic model for nucleotide metabolism in mitochondrial matrix which will take into account the activity of nucleoside diphosphate kinase in the matrix space, the probability that the GTP:GDP and ATP:ADP ratios are maintained at different values, and the sources and disposition of GTP in mitochondrial matrix reactions. The mitochondrion is the subcellular organelle of energy metabolism. This "energy factory" plays a crucial role in cell metabolism by providing energy for all vital functions. It is surprising that after several decades of intensive research, a key question still exists about how one pool of metabolites is regulated. This research applies to new technologies of measuring individual enzymes and reactants to answer the remaining questions of regulation.
