Study of gonad-stimulating substance (GSS) and 1-methyladenine which are related to starfish oocyte maturation

1.糖蛋白-激素受体家族的cDNA克隆根据膜糖蛋白激素（LH、FSH和TSH）受体的保守序列设计简并引物，从海星Asterina pectinifera卵巢中克隆了糖蛋白-激素受体家族的cDNA（apGPHR）。然而，部分纯化的性腺刺激物质（GSS）不能增加表达apGPHR的COS-7细胞中cAMP的水平，这表明海星中除了GSS之外还有一种新的神经肽是apGPHR的配体. 1-1-甲基腺嘌呤（1-MeA）是由ATP和S-腺苷甲硫氨酸（SAM）合成的。在无细胞系统中，使用卵泡细胞匀浆与缓冲溶液中，1-MeA也产生ATP和SAM在环AMP的存在下，以浓度依赖性的方式。可能是一种独特的甲基转移酶被环AMP激活产生1-MeA，尽管目前还不清楚ATP是否直接或间接被SAM甲基化。1-甲基腺嘌呤受体新合成了10个N1取代腺嘌呤类似物，用于分析1-MeA与卵母细胞表面受体的相互作用。这表明，腺嘌呤的N1-位点的区域特异性结构是重要的1-MeA受体相互作用。此外，在分子结构上，腺嘌呤的N1位带负电荷是与受体结合所必需的。其它：在卵泡细胞表面存在高活性的外ATP二磷酸水解酶（ATPDase）。外ATP酶不太可能直接参与1-MeA的产生，但外ATP酶产生的腺苷和AMP负责防止卵母细胞成熟前的早熟。

1. cDNA cloning of glycoprotein-hormone receptor familyUsing degenerate primers derived from consensus sequences of the memmalian glycoprotein hormone (LH, FSH and TSH) receptors, we cloned cDNAs (apGPHR) from ovaries of the starfish Asterina pectinifera. However, a partially purified gonad-stimulating substance (GSS) failed to increase cyclic AMP levels in COS-7 cells expressing apGPHR.This suggests that a new neural petide other than GSS is a ligand for the apGPHR in starfish.2. 1-Methyladenine biosynthesis and its regulationPrevious in vitro studies have shown that 1-methyladenine (1-MeA) is produced from ATP and S-adenosylmethionine (SAM). In a cell-free system using follicle cells homogenized with a buffer solution, 1-MeA was also produced from ATP and SAM in the presence of cyclic AMP in a concentration dependent manner. It is possible that a unique methyltransferase is activated by cyclic AMP to produce 1-MeA, although it is unclear whether ATP is methylated directly or indirectly by SAM.3. 1-Methyladenine receptorTen N1-substituted adenines as 1-MeA analogues were newly synthesized and used for analysis of the interaction between 1-MeA and its receptors on the surface of oocytes. It was suggested that regional specific structures of the N1-site of adenine are important to interact with 1-MeA receptors. In addition, on the molecular structure a negative charge in N1-site of adenine is required for the binding of the receptors.Others : A high activity of ecto-ATP diphosphohydrolase (ATPDase) was present on the surface of ovarian follicle cells. It is unlikely that ecto-ATPDase is directly involved in 1-MeA production, but adenosine and AMP produced by ecto-ATPDase is responsible for prevention of precocity before oocyte maturation stage.