TISSUE STORES OF FOLATE: DIETARY CONTROL AND ASSAY

本实验室发现了四种叶酸结合蛋白（fbp）

This laboratory has discovered four folate binding proteins (FBPs) which are enzymes that carry out previously unknown functions of the folate coenzymes. One is glycine N-methyltransferase (GNMT), an enzyme which catalyzes the methylation of glycine by S-adenosylmethionine (SAM) to form sarcosine and S-adenosylhomocysteine (SAH). Native GNMT contains bound 5- methyltetrahydrofolate (5-CH3-THF) as an allosteric inhibitor. It has been proposed that GNMT controls SAM levels in the cell by linking transmethylation to the de novo synthesis of methyl groups. Rats fed methyl deficient diets have a high incidence of spontaneous liver cancer. The proposed role for GNMT predicted that its activity should be decreased in animals fed these diets. Previous studies showed that GNMT activity was indeed lower as predicted and that total liver folate was also markedly (75%) lower, but the reasons for these changes were inconclusive. One aim of the folate coenzymes. Two other aims will investigate the mechanism of the reduced GNMT activity using whole animals and hepatocytes. GNMT is also very abundant in pancreatic acini. Pancreatic GNMT has tightly bound folate suggesting that methylation may play an important role in acinar cell function. This is consistent with the well recognized inhibition of pancreatic secretion by ethionine, a specific inhibitor of biological methylation. A fourth specific aim is thus to characterize pancreatic GNMT and identify the site at which ethionine blocks the stimulus-secretion pathway of the pancreas. One of the cytosolic FBPs is the bifunctional enzyme 10-formyl-THF dehydrogenase/hydrolase. Recently the bifunctional nature of the enzyme was challenged by contaminant. The fifth specific aim is to clone the cDNA for 10-formyl-THF dehydrogenase/hydrolase. Recently the bifunctional nature of the enzyme was challenged by studies which found only dehydrogenase activity; hydrolase activity being ascribed to a contaminant. The fifth specific aim is to clone the cDNA for 10-formyl-THF dehydrogenase/hydrolase and express the protein in high yield. This will permit the absolute determination of whether the enzyme has one or both activities. Previous work has shown that native GNMT contains two moles of phosphate per tetramer and that phosphorylation by cAMP-dependent protein kinase increases GNMT activity. The last specific aim will be to clone the cDNA for GNMT and express it in a bacterial system to provide a source of unphosphorylated enzyme.