FOLATE, HOMOCYSTEINE AND METHYL GROUP METABOLISM

甘氨酸N-甲基转移酶(GNMT)是一种存在于肝脏胞浆和胰腺外分泌物中的四聚体酶，最初作为叶酸结合蛋白被分离出来。它维持S-腺苷蛋氨酸和S-腺苷同型半胱氨酸的比例，后者调节所有的甲基化反应。甲基团的从头合成需要叶酸。甲基代谢和胰腺外分泌之间的关系已经知道很多年了。我们先前在体内已经证明，叶酸缺乏会降低SAM/SAH比率，并抑制胰腺外分泌。我们还表明，提高AR42J胰腺外分泌细胞株SAH水平的处理抑制了分泌。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有效的外分泌抑制物。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是分离参与外分泌的甲基化中间体(S)，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的过程中发挥了一种新的非酶作用。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，将四聚体酶解离为单体。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是由胞浆中的四聚体酶形成的，它是能够进入细胞核的单体。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.