The long term objective of this proposal is to clarify the metabolic role of the intracellular folate binding proteins (FBPs). Three of the four FBPs in rat liver are enzymes which cary out unusual reactions involving folate coenzymes. One FBP in the cytosol is glycine N-methyltransferase (GNMT) which carries out the S-adenosylmethionine (SAM) dependent methylation of glycine to form sarcosine and S-adenosylhomocysteine (SAH). GNMT activity is inhibited when folate is bound and this enzyme may help maintain the ratio of SAM to SAH when dietary methionione is limiting. Rats fed methyl deficient diets show a high incidence of hepatic tumors and have an altered ratio of SAM to SAH. Hepatocytes from normal and methyl group deficient rats will be used to measure the flux of methyl groups form methionine to methylated metabolites under conditions which perturb the levels of folate and vitamin B12. This will be compared with levels of GNMT activity. Alloxan diabetes produces elevated GNMT activity. Experiments will examine whether this is a response to gluconeogenesis and whether there is elevated enzyme protein. The mitochondrial FBPs are two enzymes, dimethylglycine dehydrogenase and sarcosine dehydrogenase. Tightly bound H4 folate polyglutamate serves to accept an hydroxymethyl group produced in these reactions. Studies will be done to determine whether these enzymes form a complex with mitochondrial serine hydroxymethyltransferase. This would permit """"""""channeling"""""""" and a more efficient use of this vitamin. Cross-linking reagents will be used to identify complexes formed in vitro and in vivo within the mitochondria. A second FBP from the cytosol has no known function. It may participate in the conversion of 5-methyl-thioadenosine (MTA) to methionine in the polyamine pathway which involves the production of a one-carbon unit. Nutritional studies using both rats and tissue culture will be used to determine whether the conversion of MTA to methionine is folate dependent or requires the FBP. Immunochemical methods will be used to measure the tissue distribution of this cytosolic FBP. Purification and characterization of the intracellular FBPs from human liver will be started. Techniques used in the purification of these proteins in rat liver will be adapted to the human tissue. These studies will clarify the role of the FBPs in human metabolism and nutrition.
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