With mitochondria, in conjunction with energetically manipulated steady-state cell-free systems, we propose to evaluate, with the aid of a procedure for continuous monitoring of the membrane potential (delta psi) various relationships relevant to the basic mechanism of the energy-transducing system: e.g. changes in respiration, phosphorylation of ADP, the phosphorylation potential, deltapH and redox state resulting from experimental perturbation of energy load (ATPase or a protonophore). These parameters will be evaluated together with carbon and nitrogen flux measurements in several model systems. The hypothesis is that mitochondrial functional characteristics are altered at the level of altered efficiency of energy transduction in altered endocrine states and in malignancy. Several models in these reconstituted systems will be investigated: (a) the nature of the reported effects of Ca2+ on the above parameters; (b) the nature of the effects of acute glucagon treatment on energy transduction by liver mitochondria; (c) the nature of hypo- and hyperthyroidism on mitochondrial bioenergetics; (d) liver mitochondria from hypercholesterolemia animals and from hepatomas have elevated cholesterol content in their inner membrane. We will alter mitochondrial cholesterol content with a new in vitro procedure for use in this multifaceted bioenergetic and flux-measured system; and (e) the complete urea cycle will be reconstituted in a cell-free system to evaluate lesions or alterations in this process in liver disease. Interorgan relationships in the disposition of carbon and nitrogen principally between muscle and liver is a subject of on-going and proposed investigations. Specifically, (a) we have recently proposed that large amounts of partial degradation products of the branched-chain amino acids are actually end products released from muscle, we will evaluate the capacity of liver to metabolize these putative intermediates to glucose and CO2, using isolated hepatocytes; and (b) we have recently shown that muscle catabolizes methionine (also partially) by an uncharacterized pathway, with the use of perfused muscle and, subsequently with purified cell components, to describe this pathway and its control. Endocrine and dietary effects on these systems will be evaluated. These, and the cell-free systems described, provide a framework at several levels of organization, to evaluate intracellular, intra- and interorgan metabolic fluxes under simulated physiological and pathological conditions.
