FOLATE, HOMOCYSTEINE AND METHYL GROUP METABOLISM

甘氨酸n -甲基转移酶（Glycine N-methyltransferase， GNMT）作为叶酸结合蛋白首次被分离出来，是存在于肝细胞质和胰腺外分泌的一种丰富的四聚体酶。它维持S-腺苷蛋氨酸（SAM）与S-腺苷同型半胱氨酸的比例，调节所有甲基化反应。叶酸是甲基从头合成所必需的。甲基代谢与胰腺外分泌之间的关系已被发现多年。我们之前的研究表明，叶酸缺乏会降低SAM/SAH比率，并抑制体内胰腺外分泌。我们还发现，升高AR42J胰腺外分泌细胞系SAH水平的治疗会抑制分泌。小g蛋白的c端以可逆的方式羧基甲基化，我们还发现g蛋白c端结构类似物是外分泌的有效抑制剂。我们的第一个假设是，SAH水平的升高抑制了外分泌所必需的小G蛋白的甲基化。具体目的1是分离可能参与外分泌的甲基化中间体，并确定叶酸缺乏对其形成的影响。最近在另一个实验室的研究表明，GNMT亚基作为某些多环芳烃的受体在诱导细胞色素P450中具有新的非酶作用。我们已经证明，荧光素标记的GNMT通过修饰用于亚基相互作用的关键赖氨酸将四聚体酶解离成单体。单体形式迅速进入细胞核，与染色质（可能是DNA）结合。我们的第二个假设是，细胞质溶胶中的四聚体酶形成了少量的单体GNMT，它是可以进入细胞核的单体。具体目标2是确定负责四聚体GNMT解离的因素，并确定解离的GNMT结合到DNA的哪个区域。

Glycine N-methyltransferase (GNMT), first isolated as a folate binding protein, is an abundant tetrameric enzyme in liver cytosol and the exocrine pancreas. It maintains the ratio of S- adenosylmethionine (SAM) to S-adenosylhomocysteine which regulates all methylation reactions. Folate is needed for the de novo synthesis of methyl groups. A relationship between methyl group metabolism and pancreatic exocrine secretion has been known for many years. We have previously shown that folate deficiency reduces the SAM/SAH ratio and inhibits pancreatic exocrine secretion in vivo. We also showed that treatments which elevated SAH levels in the AR42J pancreatic exocrine cell line inhibited secretion. The C-terminus of small G-proteins is carboxylmethylated in a reversible manner and we also showed that structural analogues of the G-protein C-terminus are potent inhibitors of exocrine secretion. Our first hypothesis is that elevation of SAH levels inhibits methylation of a small G protein necessary for exocrine secretion. Specific Aim 1 is to isolate the putative methylated intermediate(s) involved in exocrine secretion and determine the effects of folate deficiency on their formation. Recent studies in another laboratory have suggested a novel non-enzymatic role for GNMT subunits as a receptor for certain polycyclic aromatic hydrocarbons in the induction of cytochrome P450. We have shown that fluorescein-labeled GNMT dissociates the tetrameric enzyme into monomers by modifying critical lysines that are used in subunit interaction. The monomeric form rapidly enters the nucleus where it binds to chromatin, probably DNA. Our second hypothesis is that a small amount of monomeric GNMT is formed from the tetrameric enzyme in the cytosol and it is the monomer which can enter the nucleus. Specific Aim 2 is to determine the factors responsible for dissociation of tetrameric GNMT and determine to which region of DNA the dissociated GNMT is bound.