Photolabeling of alcohol binding sites L1:

描述（由申请人提供）：我们的总体假设是乙醇诱导的细胞粘附抑制是L1细胞粘附分子上的酒精结合位点介导的，这是发育中的神经系统损伤的一个可能原因。不同醇对细胞粘附的抑制（激动剂作用）表现出显著的结构特异性。其他醇（例如，1-辛醇）非竞争性地拮抗乙醇对l介导的细胞粘附和小鼠全胚胎培养发育的影响。我们假设这些位点在L1上，因为L1突变儿童的大脑与胎儿酒精谱系障碍（FASD）儿童的大脑相似。该建议的新颖之处在于使用最近开发的光亲和性醇类似物，3-叠氮丁醇（一种激动剂）和3-叠氮醇（一种拮抗剂）分别对激动剂和拮抗剂结合位点进行光标记。Specific Aim 1通过用3-azibutanol对L1粘附分子上存在酒精激动剂位点进行光标记来验证这一假设，3-azibutanol对细胞粘附的作用类似于乙醇。用[3H]3-azibutanol光标记纯化L1，用高效液相色谱法（HPLC）分离含光乙醇的片段。光掺入的化学计量学将用质谱法测定。此外，目标片段的消化将产生适合质谱测序的片段。为了评估药理学相关性，将确定每个光结合位点的表观解离常数和药理学，并将其与平行细胞粘附实验所得的结果进行比较。特异性目的2类似地测试了L1上存在单独的酒精拮抗剂位点的假设，使用光亲和标记3-azioctanol，它在微摩尔浓度下抑制乙醇诱导的细胞粘附抑制。我们期望在LI上每个药理学特征良好的位点的结合口袋中定义至少一个氨基酸。分别确定激动剂和拮抗剂位点将有助于了解FASD的分子基础，并加速开发阻断乙醇对发育中的神经系统毒性作用的药物。

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.