PROPERTIES OF DYNEIN ISOENZYMES

动力蛋白同工酶的特性

基本信息

批准号：
2900556
负责人：
IAN R GIBBONS
金额：
$ 12.39万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
1982
资助国家：
美国
起止时间：
1982-04-01 至 2002-03-31
项目状态：
已结题

项目摘要

The broad long-term objectives of this project are (i) to determine the structural, chemical and enzymatic properties of outer-arm dynein, including its interaction with tubulin, and to relate these properties to the role of the arms in producing flagellar and ciliary motility, (ii) to compare the properties of the other isoforms of axonemal dynein to those of outer-arm dynein and to relate the differences to their respective roles in axonemal beating, (iii) to study the differences between cytoplasmic and axonemal dyneins and to relate them to their roles in cell motility. In the up-coming five-year project period much of the effort will be concentrated on using a combination of protein chemistry and molecular biology to follow up the various leads that have been opened by our recent sequencing of the Beta heavy chain of outer arm dynein from sea urchin embryos. Photoaffinity labeling will be used to identify the several candidate nucleotide-binding sites revealed in the amino acid sequence. Localized regions of conserved amino acid sequence that span the hydrolytic ATP-binding site will be used to amplify and sequence the corresponding 400 base pair regions of other dynein isoforms from sea urchin embryos and testis, as well as from selected tissues of Drosophila and mouse. By grouping the isoforms into families according to their degree of homology to each other and to the Beta heavy chain and then correlating the families with their tissue of origin, it should be possible to distinguish axonemal and cytoplasmic dyneins, and to identify the functional equivalents of the various axonemal dynein isoforms in different species. As this grouping into families proceeds, the sequence of selected isoforms will be extended in order to look for regions of conserved sequence that are further removed form the hydrolytic ATP-binding site and that show homology to the microtubule-binding sites of the other microtubule-associated proteins whose sequence is known. Selected conserved regions of the Beta chain will be expressed in E coli and examined in order to determine whether the expressed protein shows ATP-sensitive or ATP-insensitive microtubule-binding properties. The cellular localization of the various dynein isoforms will be examined with poly-clonal antibodies to expressed peptides representing unique regions of their sequence. As the project progresses, attempts will be made to express functional domains of the Beta chain that retain its capability to translocate microtubules over glass. The results of this study are expected to clarify the fundamental mechanisms by which dynein produces transport along microtubules in mitosis and in nerve axons, and so to contribute to understanding the many problems associated with improper chromosomal segregation and the lack of nerve regeneration after injury.

该项目的广泛长期目标是（i）确定外臂动力蛋白的结构，化学和酶促特性，包括其与微管蛋白的相互作用，并关联这些特性掌握武器在产生鞭毛和睫状运动中的作用，（ii）比较轴突动力蛋白的其他同工型的特性到外臂动力蛋白的那些，并将差异与它们的差异联系起来在轴突跳动中各自的作用，（iii）研究差异在细胞质和轴突动力蛋白之间细胞运动中的作用。在即将上映的五年项目期间努力将集中于使用蛋白质的组合化学和分子生物学，以跟进具有我们最近对Beta重链外部的测序打开了来自海胆胚胎的手臂动力蛋白。光性标签将使用确定几个在氨基酸序列。保守氨基酸的局部区域跨越水解ATP结合位点的序列将用于放大和对相应的400个基对区域进行序列海胆胚胎和睾丸的动力蛋白同工型，以及果蝇和小鼠的选定组织。通过将同工型分组为家庭根据他们的同源程度，彼此的同源性程度 Beta重链，然后将家庭与他们的组织相关联起源，应该有可能区分轴突和细胞质动力蛋白，并确定各种功能等效物不同物种中的轴突动力蛋白同工型。当这个分组家庭进行，选定的同工型的顺序将扩展为了寻找进一步删除的保守序列区域形成水解ATP结合位点，并与其他微管相关蛋白的微管结合位点其序列是已知的。 β链的选定保守区域将在E大肠杆菌中表达并检查以确定是否是否表达的蛋白质显示对ATP敏感或不敏感的微管结合特性。细胞定位各种动力蛋白同工型将使用多旋抗体进行检查表示代表其序列独特区域的肽。作为该项目进展，将尝试表达功能保留其易位能力的Beta链的域玻璃上的微管。这项研究的结果有望阐明动力蛋白产生运输的基本机制沿着有丝分裂和神经轴突中的微管，因此有助于了解与染色体不当相关的许多问题受伤后的隔离和缺乏神经再生。