REGULATION OF THIAMINE-DEPENDENT ENZYMES INVOLVED IN GLUCOSE

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Chronic heavy alcohol drinking leads to malnutrition often associated with losses of body weight and other functions. This is mainly due to aberrant metabolism of glucose which normally provides energy needed for maintaining normal physiological functions. It was reported that one of the leading candidates for these changes might be due to deficiency of thiamine, vitamin B, which is a cofactor for several key enzymes involved in glucose metabolism. These are pyruvate dehydrognease, alpha- ketoglutarate dehydrogenase, and transketolase. The activities of these enzymes were altered (reduced) in several disease states often associated with alcohol abuse. Despite numerous reports on the biochemical properties, the structures and molecular regulation of these enzymes have not been well investigated. Because of the importance of these enzymes in energy metabolism, the molecular mechanism of the regulation of these enzymes was studied. Mitochondrial pyruvate dehydrogenase and alpha- ketoglutarate dehydrogenase and cytosolic transketolase were purified to homogeneity and specific antibodies were generated in rabbits. These purified proteins were subjected to N-terminal and internal amino acid sequencing analyses for the generation of oligodeoxynucleotides needed for cDNA cloning for these enzymes. Using polyclonal antibodies and oligodeoxynucleotides as probes, cDNA clones for these enzymes were identified and characterized. Our results indicated that the amounts of immunoreactive enzymes are decreased in brain of thiamine deficient rats. The mechanism of the reduction in these enzymes is being studied by molecular biological approaches.