A deficiency of folic acid leads to a life-threatening megaloblastic anemia. Vitamin B12 deficiency and exposure to the anesthetic gas, nitrous oxide (N2O), causes a functional folate deficiency because folate coenzymes, needed for DNA and RNA synthesis, are trapped as 5- methyltetrahydrofolate. This functional deficiency leads to megaloblastic anemia and to neuropathy. There is increasing evidence that one-carbon metabolism is involved in the exocrine function of the pancreas and that alcohol perturbs pancreatic one-carbon metabolism. Folates in the liver are partitioned between the cytosolic compartment and the mitochondrial compartment of the cell. The long term objectives of this proposal is to delineate the compartmentation of one-carbon metabolites in liver and pancreas. This will be accomplished by measuring the concentrations of the individual folate coenzymes in cytosolic and mitochondrial fractions from liver and pancreas. The effects of N2O exposure, of acute and chronic ethanol, and of folate deficiency on folate coenzyme compartmentation and on the concentrations of S-adenosylmethionine and S-adenosylhomocysteine will be determined in pancreas. Various enzymes involved in one-carbon metabolism will be assayed in liver and pancreas to help explain the observed effects of these conditions. The mechanism of transport of folates into pancreatic acinar cells will be determined. The effects of ethanol consumption on acinar cell folate transport and on transport of folates across the liver basolateral membrane will be studied. Mitochondria contain a carrier-mediated system for transporting the reduced, naturally occurring folate derivatives. There is evidence that this carrier may be covalently labeled with 5-formyltetrahydrofolate. This will aid in purification of the carrier by 2-dimensional electrophoresis or conventional chromatographic means. Amino acid sequences from the carrier protein will provide information necessary to synthesize DNA probes for use in cloning the carrier cDNA. The clone will be sequenced by the dideoxynucleotide method and the derived amino acid sequence determined. Similar techniques will be employed to purify and clone the carrier protein from pancreatic acinar cells. The cDNA and antibodies to the carriers will be used to determine if dietary factors (e.g., folate deficiency) and alcohol up- or down regulate the expression of the carriers, in vivo.
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