Deficiencies of carnitine and of the carnitine acyltransferases have been reported in a variety of clinical conditions. The role of carnitine in shuttling activated long chain acyl groups into mitochondria for beta-oxidation is well understood, but other roles for this ubiquitous and plentiful compound are only now being investigated. Although physical effects on membranes may yet prove important, the major intracellular metabolic role of carnitune is to buffer the acylation state of the small amount of CoA. This proposal will investigate how this is achieved. The reversible equilibrium (buffering) is mediated by the carnitine acyltransferases and is affected by compounds regulating their activity, such as malonyl-CoA which regulates flux through beta-oxidation by inhibiting the generation of acyl-carnitine on the mitochondrial membrane. This proposal covers experiments to characterize the five types of acyltransferases both in purified form and within the native membrane of each using established kinetic and protein chemistry techniques. The data should yield comparisons of the kinetic mechanism of each, of the effects of the membrane environment on the acyl-chain length substrate specificity, and of how malonyl-CoA and other inhibitbors exert their effect. The basis for further insight will be sought by expressing the cloned gene for the peroxisomal carnitine acyltransferase in yeast to produce large quantities of the enzyme both in its native form and in forms modified by standard molecular biology techniques. Immunohistological techniques will be used to identify the peroxisomal enzyme in human and rat tissue slices to locate the enzyme and to probe for deficiencies without the need for fresh tissue samples. The relationship between peroxisomal oxidation and transferases will be explored and compared also in various brain regions. In brain, roles outside transport for A-oxidation are likely to be important but are as yet unknown. One possibility is the buffering of acyl-CoA levels available for repair of membrane lipids. This role, first demonstrated in erythrocyte membranes is important for repair of oxidative damage to membranes and for the integration of that process with fatty acid availability and the catabolic needs of the cell.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Medical Biochemistry Study Section (MEDB)
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University of California San Francisco
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