TISSUE STORES OF FOLATE: DIETARY CONTROL AND ASSAY

本实验室发现了四种叶酸结合蛋白(FBPs)，它们 是执行以前未知的叶酸功能的酶 辅酶。一种是甘氨酸N-甲基转移酶(GNMT)，一种可以 S-腺苷甲硫氨酸催化甘氨酸甲基化生成 肌氨酸和S-腺苷同型半胱氨酸。本机GNMT包含绑定的5- 甲基四氢叶酸(5-CH3-THF)作为变构抑制剂。一直以来 提出GNMT通过链接来控制细胞内的SAM水平 甲基化转变为甲基从头合成。喂食的大鼠 缺乏甲基的饮食有很高的自发性肝癌发病率。 GNMT的拟议作用预计其活动应减少。 在喂食这些食物的动物身上。先前的研究表明，GNMT的活动是 确实如预测的那样低，肝脏总叶酸也明显 (75%)下降，但这些变化的原因尚不确定。 叶酸辅酶的一个目的。另外两个目标将调查 用全动物和肝细胞研究GNMT活性降低的机制。 GNMT在胰腺腺泡内也非常丰富。胰腺GNMT有 紧密结合的叶酸表明甲基化可能起着重要作用 在腺泡细胞功能方面。这与公认的 乙硫氨酸对胰腺分泌的抑制作用 生物甲基化。因此，第四个具体目标是将 胰腺GNMT并确定乙硫氨酸阻断 胰腺的刺激分泌途径。 胞质FBPs之一是双功能酶10-甲酰-四氢呋喃 脱氢酶/水解酶。近年来，酶的双功能性质 受到了污染物的挑战。第五个特定目标是克隆cdna。 对于10-甲酰-四氢呋喃脱氢酶/水解酶。最近，两种功能 这种酶的性质受到了研究的挑战，研究发现只有 脱氢酶活性；水解酶活性归因于污染物。 第五个目标是克隆10-甲酰-四氢呋喃的cdna。 脱氢酶/水解酶，并高效表达。这将是 允许绝对确定酶是否有一种或两种都有 活动。 先前的工作表明，天然GNMT含有两个摩尔的磷酸盐 每四聚体和cAMP依赖的蛋白激酶的磷酸化 增加GNMT活动。最后一个具体目标是克隆cdna。 并在细菌系统中表达，以提供来源 非磷酸化的酶。

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.