FOLATE, HOMOCYSTEINE AND METHYL GROUP METABOLISM

甘氨酸 N-甲基转移酶 (GNMT) 最初作为叶酸结合蛋白被分离出来，是肝细胞质和外分泌胰腺中丰富的四聚体酶。 它维持 S-腺苷甲硫氨酸 (SAM) 与 S-腺苷高半胱氨酸的比例，调节所有甲基化反应。 甲基的从头合成需要叶酸。 多年来，甲基代谢与胰腺外分泌之间的关系已为人所知。 我们之前已经证明，叶酸缺乏会降低 SAM/SAH 比率并抑制体内胰腺外分泌分泌。 我们还表明，提高 AR42J 胰腺外分泌细胞系中 SAH 水平的治疗会抑制分泌。 小G蛋白的C末端以可逆的方式羧甲基化，我们还表明G蛋白C末端的结构类似物是外分泌分泌的有效抑制剂。 我们的第一个假设是 SAH 水平升高会抑制外分泌分泌所需的小 G 蛋白的甲基化。 具体目标 1 是分离参与外分泌分泌的推定甲基化中间体，并确定叶酸缺乏对其形成的影响。 另一个实验室最近的研究表明，GNMT 亚基作为某些多环芳烃的受体在细胞色素 P450 的诱导中具有一种新的非酶促作用。 我们已经证明，荧光素标记的 GNMT 通过修饰亚基相互作用中使用的关键赖氨酸，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，少量的单体GNMT是由胞浆中的四聚体酶形成的，并且它是可以进入细胞核的单体。具体目标 2 是确定导致四聚体 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.