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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK013939-19
Application #
3225172
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1977-04-01
Project End
1993-03-31
Budget Start
1990-04-01
Budget End
1991-03-31
Support Year
19
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Type
Schools of Medicine
DUNS #
005436803
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Schild, L; Blair, P V; Davis, W I et al. (1999) Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels. Biochim Biophys Acta 1413:14-20
Dietzen, D J; Davis, E J (1994) Excess membrane cholesterol is not responsible for metabolic and bioenergetic changes in AS-30D hepatoma mitochondria. Arch Biochem Biophys 309:341-7
Dietzen, D J; Davis, E J (1993) Oxidation of pyruvate, malate, citrate, and cytosolic reducing equivalents by AS-30D hepatoma mitochondria. Arch Biochem Biophys 305:91-102
Davis, E J; Davis-van Thienen, W I (1991) An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation. Arch Biochem Biophys 289:184-6
Kunz, W S; Davis, E J (1991) Control of reversible intracellular transfer of reducing potential. Arch Biochem Biophys 284:40-6
Davis, E J; Davis-van Thienen, W I (1989) Force-flow and back-pressure relationships in mitochondrial energy transduction: an examination of extended state 3-state 4 transitions. Arch Biochem Biophys 275:449-58
Scislowski, P W; Bremer, J; van Thienen, W I et al. (1989) Heart mitochondria metabolize 3-methylthiopropionate to CO2 and methanethiol. Arch Biochem Biophys 273:602-5