This application is a request for an ADAMHA Research Scientist Development Award/Level II for William S. Thayer, Ph.D. The award will allow Dr. Thayer to devote his full efforts to research, and will enhance his scientific capabilities by allowing him to broaden his experience in alcohol-related research and learn the use of new experimental techniques. The latter will include immunochemistry and nuclear magnetic resonance spectroscopy. The proposed research will study the effects of ethanol and ethanol consumption on mitochondrial membrane assembly and mitochondrial bioenergetics. Rats fed ethanol chronically will be used as an animal model for alcoholic fatty liver. Isolated hepatocytes, liver mitochondria, phosphorylating submitochondrial particles, and phospholipid vesicles reconstituted with specific mitochondrial proteins, particularly cytochrome oxidase, will be used as experimental systems. An inactive form of cytochrome oxidase, recently identified by immunochemical techniques in submitochondrial particles from alcoholic rats, will be isolated and characterized. The biological turnover of heme and protein components of cytochrome oxidase, and other mitochondrial membrane proteins, in vivo will be determined from time courses of decreases in radioactivity following labeling with specific precursors. Effects of alcohol on the synthesis and assembly of cytochrome oxidase, and other mitochondrial membrane proteins, will be investigated by immunochemical techniques using isolated hepatocytes. Reconstitution of mitochondrial membrane proteins into phospholipid vesicles will be studied as a model system for membrane assembly. The role of lipid composition and physical properties in determining the protein content and enzymatic activity of reconstituted vesicles will be explored. Effects of ethanol consumption on mitochondrial bioenergetics and function in the liver cell will be studied by examining the regulatory relationships between respiration rate, electrochemical proton gradient and the adenine nucleotide phosphorylation system. Effects of ethanol on proton translocation reactions involved in generation of the electrochemical proton gradient will also be investigated. These studies will contribute to an understanding of the bioenergetic consequences of alcohol consumption and will provide insights concerning fundamental mechanisms of ethanol toxicity.
