Photolabeling of alcohol binding sites L1:

描述(由申请人提供)：我们的总体假设是，酒精诱导的细胞黏附抑制是由L1细胞黏附分子上的酒精结合位点介导的，这是发育中的神经系统受损的一个可能原因。不同醇类对乙醇抑制细胞黏附(激动剂作用)表现出显著的结构特异性。其他醇(如正辛醇)非竞争性拮抗乙醇对LL介导的细胞黏附和小鼠全胚胎培养的影响。我们假设这些位点在L1上，因为L1突变儿童的大脑与患有胎儿酒精谱系障碍(FASD)的儿童的大脑相似。这一建议的新颖之处在于使用最近开发的光亲和醇类似物，3-氮杂丁醇(激动剂)和3-氮杂辛醇(拮抗剂)分别光标记激动剂和拮抗剂的结合部位。特殊目的1通过用3-氮杂丁醇对L1黏附分子进行光标记来检验这一假设，3-氮杂丁醇在细胞黏附中的作用类似于乙醇。用[~3H]3-氮杂丁醇对纯化的L1进行光标记物的消化，用高效液相色谱分离光合乙醇碎片。光掺入的化学计量比将通过质谱学进行测量。此外，对目标片段的消化将产生适合于通过质谱学进行测序的片段。为了评估药理学相关性，将测定每个光结合部位的表观解离常数和药理学，并将其与平行细胞黏附实验获得的结果进行比较。特殊目的2类似地使用光亲和标记3-氮杂辛醇来测试假设，即在L1上有单独的酒精拮抗剂位点，它在微摩尔浓度下抑制乙醇诱导的细胞黏附抑制。我们希望在LI上每个药理特征良好的位点的结合口袋中定义至少一个氨基酸。激动剂和拮抗剂位置的确定将分别有助于了解FASD的分子基础，并加快药物的开发，以阻断乙醇对发育中的神经系统的毒性影响。

DESCRIPTION (provided by applicant): Our overall hypothesis is that ethanol-induced inhibition of cell adhesion, one possible cause of damage in the developing neural system, is mediated by alcohol binding sites on the L1 cell adhesion molecule. Different alcohols show remarkable structural specificity for alcohol inhibition of cell adhesion (agonist action). Other alcohols (e.g., 1-octanol) noncompetitively antagonize the effects of ethanol on Ll-mediated cell adhesion and on the development of mouse whole embryo cultures. We hypothesize that these sites are on L1 because the brains of children with L1 mutations resemble those of children with fetal alcohol spectrum disorder (FASD). The novel aspect of this proposal is the use of recently developed photoaffinity alcohol analogs, 3-azibutanol (an agonist) and 3-azioctanol (an antagonist) to photolabel the agonist and antagonist binding sites respectively. Specific Aim 1 tests the hypothesis that there are alcohol agonist sites on the L1 adhesion molecule by photolabeling them with 3-azibutanol, which acts similarly to ethanol on cell adhesion. Purified L1 photolabeled with [3H]3-azibutanol will be digested and fragments photoincorporating alcohol separated by HPLC. The stoichiometry of photoincorporation will be measured by mass spectrometry. Further, digestion of target fragments will produce fragments suitable for sequencing by mass spectrometry. To assess pharmacological relevance, the apparent dissociation constant and pharmacology of each photoincorporation site will be determined and compared to those obtained from parallel cell adhesion experiments. Specific Aim 2 similarly tests the hypothesis that there are separate alcohol antagonist sites on L1 using the photoaffinity label, 3-azioctanol, which inhibits ethanol-induced inhibition of cell adhesion at micromolar concentrations. We expect to define at least one amino acid in the binding pocket of each pharmacologically well-characterized site on LI. Identification of the agonist and of the antagonist site will, respectively, aid in understanding the molecular basis for FASD and accelerate the development of drugs that block the toxic effects of ethanol on the developing nervous system.