TISSUE STORES OF FOLATE: DIETARY CONTROL AND ASSAY

该实验室发现了四种叶酸结合蛋白（FBPs）， 是一种酶，它执行叶酸以前未知的功能， 辅酶 一种是甘氨酸N-甲基转移酶（GNMT）， 催化甘氨酸被S-腺苷甲硫氨酸（SAM）甲基化，形成 肌氨酸和S-腺苷高半胱氨酸（SAH）。 天然GNMT含有结合的5- 甲基四氢叶酸（5-CH 3-THF）作为变构抑制剂。 已经 提出GNMT通过连接细胞中的SAM水平来控制 从甲基转移到甲基的从头合成。 大鼠 缺乏甲基的饮食具有自发性肝癌的高发病率。 GNMT的拟议作用预测其活性应降低 在这些食物喂养的动物中。 以前的研究表明，GNMT活性是 确实如预测的那样降低了，而且肝脏中的总叶酸也显著地 (75%）较低，但这些变化的原因尚不确定。 叶酸辅酶的作用之一。 另外两个目标将调查 使用整个动物和肝细胞研究GNMT活性降低的机制。 GNMT在胰腺腺泡中也非常丰富。 胰腺GNMT具有 与叶酸紧密结合，这表明甲基化可能起着重要作用， 在腺泡细胞功能中。 这与公认的 一种特异性的胰腺分泌抑制剂， 生物甲基化 因此，第四个具体目标是描述 胰腺GNMT，并确定在该网站上的Ethylene阻断 胰腺的刺激分泌途径。 胞质FBPs之一是双功能酶10-甲酰-THF 脱氢酶/水解酶。 最近，酶的双功能性质 受到污染物的挑战。 第五个具体目标是克隆cDNA 用于10-甲酰基-THF脱氢酶/水解酶。 最近，双功能 这种酶的性质受到了研究的挑战， 脱氢酶活性;水解酶活性归因于污染物。 第五个具体目标是克隆10-甲酰-THF的cDNA 脱氢酶/水解酶，并以高产率表达蛋白质。 这将 允许绝对确定酶是否具有一个或两个 活动 先前的工作表明，天然GNMT含有两摩尔磷酸盐， 和cAMP依赖性蛋白激酶磷酸化 增加GNMT活性。 最后一个具体目标是克隆cDNA 并在细菌系统中表达，以提供 非磷酸化酶

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.