STRUCTURAL DISTINCTIONS AMONG ACETYLCHOLINESTERASES

乙酰胆碱酯酶之间的结构差异

基本信息

批准号：
3396988
负责人：
TERRONE L ROSENBERRY
金额：
$ 13.26万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1980
资助国家：
美国
起止时间：
1980-07-01 至 1989-06-30
项目状态：
已结题

项目摘要

The overall objectives of our laboratory are to determine the structural and biosynthetic relationships among the several forms of acetylcholinesterase (AChE) in mammalian tissues and the ways in which these forms are biologically regulated. The predominant form of AChE in neuromuscular junctions is an assembly of catalytic subunits and collagen-like noncatalytic subunits that appear to anchor this AChE form in the extracellular basement membrane. Alternative membrane-bound forms of AChE, found in nonjunctional muscle membrane, brain and erythrocytes, are devoid of collagen-like components and are localized directly in phospholipid membranes as integral membrane proteins. Human erythrocyte AChE (RBC AChE), which appears to be a good model for other integral membrane AChEs, is an amphipathic protein whose small hydrophobic membrane-binding domain is cleaved by certain proteases. A primary specific aim of this proposal is the amino acid sequence analysis of selected regions of the RBC AChE catalytic subunits. RBC AChE can be selectively radiolabeled at 4 sites: the hydrophobic domain can be labeled by a photolabeling reagent; the N-terminal amino acid, by reductive methylation; the single sulfhydryl involved in intersubunit disulfide bonding, by selective reduction and alkylation; and the active site, by organophosphorylation. RBC AChE peptides generated by CNBr and trypsin will be fractionated by gel exclusion chromatography and HPLC. Isolated peptides, particularly those selectively labeled, will be sequenced. Sequence information about the hydrophobic domain should clarify how amphipathic AChEs interact with phospholipid membranes. In a second major specifc aim, oligoDNA sequences corresponding to AChE mRNA segments for part of the RBC AChE polypeptides will be deduced from selected peptide sequences. These oligoDNAs will be chemically synthesized and labeled for use as probes for screening cDNA libraries. Identification of cDNAs for AChE mRNAs will provide a powerful tool for examining biosynthetic relationships among AChEs and for detailing the amino acid sequence differences among these AChEs. A third aim is to isolate AChE from mammalian brain and characterize any differences in the hydrophobic domain from that of RBC AChE. A fourth aim is focused on the collagen-like tail subunits from AChE isolated from eel electric organ. Attempts to obtain specific antisera to these subunits will be made, and chemical analyses to detect endogenous glycosaminoglycan associated with these subunits will be conducted. Glycosaminoglycan if present would likely play a major role in the attachment of AChE to the neuromuscular junction.

我们实验室的总体目标是确定结构和几种形式之间的生物合成关系哺乳动物组织中的乙酰胆碱酯酶（ACHE）及其方式这些形式受到生物学调节。酸痛的主要形式神经肌肉连接是催化亚基和胶原蛋白样的非催化亚基似乎将这种疼痛形式固定在细胞外膜。替代膜结合形式的形式 ACHE，在非骨膜膜，大脑和红细胞中发现的ACHE是没有类似胶原蛋白的成分，直接位于磷脂膜作为整体膜蛋白。人红细胞 ACHE（RBC ACHE），它似乎是其他积分的好模型膜酸是一种两亲蛋白，其小疏水性膜结合结构域被某些蛋白酶裂解。主要该提案的具体目的是氨基酸序列分析 RBC ACHE催化亚基的选定区域。 RBC酸痛可以是在4个地点有选择性地放射性标记：疏水结构域可以标记通过光标记试剂； N端氨基酸，通过还原性甲基化；参与亚基间二硫化物的单一硫酸通过选择性降低和烷基化结合；和主动地点，有机磷酸化。 CNBR和胰蛋白酶产生的RBC ACHE肽将通过凝胶排除色谱和HPLC分馏。孤立肽，特别是选择性标记的肽，将进行测序。有关疏水结构域的序列信息应阐明如何两亲性疼痛与磷脂膜相互作用。在第二个大满贯指定目标，寡素序列，对应于ACHE mRNA片段 RBC ACHE多肽的一部分将从选定的肽中推导序列。这些寡头将被化学合成并标记为用作筛选cDNA文库的探针。识别CDNA的 ACHE mRNA将为检查生物合成提供强大的工具酸痛之间的关系，用于详细说明氨基酸序列这些疼痛之间的差异。第三个目标是隔离疼痛哺乳动物大脑并表征疏水结构域的任何差异从加拿大皇家银行的痛苦中。第四个目标专注于胶原蛋白的尾巴从鳗鱼电器官分离的ACHE的亚基。尝试获得将对这些亚基进行特定的抗血清，并进行化学分析检测与这些亚基相关的内源性糖胺聚糖将是实施。糖胺聚糖（如果在场）可能在 ACHE的附着在神经肌肉连接处。