FOLATE, HOMOCYSTEINE AND METHYL GROUP METABOLISM

甘氨酸N-甲基转移酶（GNMT），首先作为叶酸结合蛋白被分离出来，是一种在肝细胞质和胰腺外分泌中含量丰富的四聚体酶。 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。 叶酸是从头合成甲基所必需的。 甲基代谢和胰腺外分泌之间的关系已经知道很多年了。 我们以前已经表明，叶酸缺乏降低SAM/SAH的比例，并抑制胰腺外分泌在体内。 我们还表明，在AR 42 J胰腺外分泌细胞系中升高SAH水平的处理抑制分泌。 小G蛋白的C-末端以可逆的方式被羧甲基化，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。 具体目标1是分离参与外分泌的假定甲基化中间体，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诱导细胞色素P450中具有新的非酶促作用。 我们已经表明，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，在那里它与染色质结合，可能是DNA。 我们的第二个假设是，少量的单体GNMT是由细胞质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是确定负责四聚体GNMT解离的因素，并确定解离的GNMT与DNA的哪个区域结合。

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.