Various aspects of the regulation of metabolic processes involved in energy generation in liver tissue will be investigated in the proposed research. A detailed study will be made of the mechanism and the potential effectiveness of substrate level phosphorylation derived GTP as an inhibitor of the alpha-ketoglutarate dehydrogenase (alpha- KGDH) reaction of the citric acid cycle using isolated liver mitochondria and purified alpha-KGDH from various tissues. The mechanism of the GTP inhibition of alpha-KGDH as a means of preventing the entry of reducing equivalents from all NAD-linked substrates into the electron transport chain will be investigated. The effect of elevated intramitochondrial GTP levels on the control of other reactions such as glutamate dehydrogenase which may be sensitive to this nucleotide will be studied. Second, the effectiveness of a phosphorylation-dephosphorylation mechanism for the regulation of the pyruvate dehydrogenase multienzyme complex (PDH) in intact metabolic systems will be characterized. Factors affecting the activity of the pyruvate dehydrogenase-associated protein kinase and phosphatase such as the intramitochondrial ATP and magnesium concentrations will be studied and correlated with both the activity of the PDH and the incorporation of P32 from gamma-ATP32 into the enzyme in the mitochondrial system. Also of interest will be the differential regulation of the entry of pyruvate into the citric acid cycle via the PDH reaction and/or into the gluconeogenic four-carbon pool via the pyruvate carboxylase reaction by alterations in the ATP and magnesium levels of the liver mitochondrion. Finally, a study will be made of the regulation of the flux of acetyl units either into the citric acid cycle or into the ketogenic sequence in intact isolated liver cells. Factors such as the oxidation-reduction state, the energy charge and the presence or absence of various co- substrates will be evaluated as to their effect on this system. The significance of the proposed research derives from the importance of understanding regulatory mechanisms governing metabolic energy generation and acetyl CoA metabolism in a tissue such as liver.