ACTIVE SITE DETERMINATION OF THE E-TYPE ATPASES

E 型ATP酶活性位点的测定

• 批准号: 6498963
• 负责人: TERENCE LEE KIRLEY
• 金额: $ 26.16万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6498963
• 关键词: actins; active sites; adenosine diphosphate; adenosine triphosphate; adenosinetriphosphatase; chemical binding; enzyme structure; gene expression; hydrolysis; in situ hybridization; laboratory rat; nucleotide metabolism; protein localization; protein structure function; site directed mutagenesis

The long term goal of the proposed research is to understand the structures and the functions of ecto-ATPases and ecto-apyrases in extracellular nucleotide metabolism in both normal and pathophysiological states. These enzymes, along with the related soluble apyrases, constitute the class of enzymes known as the "E-type ATPases". The E-type ATPases are important for the termination of purinergic receptor-mediated responses, including termination of responses to nucleotides used as neurotransmitters. They are also involved in cell adhesion processes, and are important for maintenance of hemostasis in the cardiovasculature. The cDNAs encoding the three human E-type ATPases will be expressed. Site directed mutagenesis will be performed to identify amino acid residues which are important for ATP and ADP hydrolysis and binding. The site-directed mutagenesis experiments will be designed based on: (1) the newly discovered homologies to the actin/heat shock protein/sugar kinase superfamily of enzymes; (2) careful analysis of amino acid residues that are conserved in all the E-type ATPases; (3) analysis of residues that are different between the ecto-ATPases and ecto-apyrases, yet conserved among the sub-types. Thus, amino acid residues important for nucleotide hydrolysis will be identified, the hypothesis that the E-type ATPases are members of the actin superfamily of proteins will be tested, and the residues important for hydrolysis of ADP in addition to ATP (i.e., residues distinguishing biochemically the ecto-apyrases from ecto-ATPases) will be identified. In addition, in situ nucleotide hybridization and immunolocalization using specific anti-peptide antibodies will be performed to learn which areas of the brain and muscle express which E-type ATPase(s), thereby establishing putative functional roles for the enzymes in extracellular nucleotide metabolism. If site-directed mutagenesis experiments continue to support the hypothesis that the E-type ATPases are members of the actin superfamily of proteins, then three dimensional computer modeling of the mutational results will be done by using the known crystal structures of members of the actin/heat shock protein/sugar kinase superfamily of proteins as templates for small segments of wild-type and mutated E-type ATPase sequences.

拟议的研究的长期目标是了解正常和病理生理状态下细胞外核苷酸代谢中的肾上腺酶和外胞酶的结构和功能。 这些酶以及相关的可溶性顶酶构成了称为“ E型ATPases”的酶类。 E型ATPases对于终止嘌呤能受体介导的反应至关重要，包括终止对用作神经递质的核苷酸的反应。 它们还参与细胞粘附过程，对于维持心血管止血物很重要。编码三种人类电子型ATPases的cDNA将被表达。 将进行定位的诱变，以鉴定对ATP和ADP水解和结合至关重要的氨基酸残基。 位置定向的诱变实验将基于：（1）酶的肌动蛋白/热激蛋白/糖激酶超家族的新发现的同源物； （2）仔细分析在所有E型ATPases中保守的氨基酸残基； （3）分析ecto-atpases和ecto-opysas之间的残基，但在亚型之间保守。 Thus, amino acid residues important for nucleotide hydrolysis will be identified, the hypothesis that the E-type ATPases are members of the actin superfamily of proteins will be tested, and the residues important for hydrolysis of ADP in addition to ATP (i.e., residues distinguishing biochemically the ecto-apyrases from ecto-ATPases) will be identified. 此外，将使用特定的抗肽抗体进行原位核苷酸杂交和免疫定位，以了解大脑和肌肉的哪个区域以及哪些E型ATPase（S），从而在细胞外核苷酸外核苷酸代谢中建立了推定的功能作用。 如果定位定向的诱变实验继续支持以下假设：蛋白质的e型ATPases是蛋白质肌动蛋白超家族的成员，那么突变结果的三维计算机建模将通过使用肌动蛋白/热休克蛋白/糖激酶超级蛋白质的蛋白质/热蛋白糖激酶超级群体的构成的晶体结构来完成序列。