SYNAPTIC VERICLE ISOLATION FROM SACCULAR HAIR CELLS

突触毛囊与囊状毛细胞的分离

• 批准号: 3424479
• 负责人: JANIE C PARK
• 金额: $ 2.44万
• 依托单位: FLORIDA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1993-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3424479
• 关键词: Anura; SDS polyacrylamide gel electrophoresis; ear hair cell; immunocytochemistry; membrane proteins; membrane reconstitution /synthesis; neurotransmitter metabolism; secretory protein; synaptic vesicles

The objective of this proposal is to develop a protocol to isolate synaptic vesicles (SVs) from frog saccular hair cells. Until a population can be successfully isolated, functional studies on SVs are severely limited. Isolated SV populations are essential for determining function and mechanism of neurotransmission, membrane synthesis, content and recycling, and enzymatic activity of SVs. In addition, the production of immunological probes for SV membrane proteins unique to hair cells requires isolated SVs. The frog saccule provides a good preliminary model for developing microisolation and purification techniques because of its relatively large size. However, the techniques to be perfected in this pilot project, and the information obtained about protein content of the SVs, should be applicable to mammalian models. In this pilot study we propose to isolate a SV-rich fraction from frog saccular hair cells by enzymatic cell separation followed by cell fractionation techniques. We will demonstrate the enrichment of vesicles in the isolated fraction by electron microscopy and immunoassay for a putative membrane protein marker, synaptophysin. We will also characterize the total protein content of the SV-rich fraction by one-dimensional SDS PAGE electrophoresis. The proposed project requires the use of microseparation techniques because of the minute quantities of tissue involved.

这项提议的目标是开发一种分离突触的方案 青蛙囊状毛细胞的囊泡(SVS)。直到一个人口能够 成功分离后，SVS的功能研究受到严重限制。 隔离的SV种群对于确定功能和 神经传递、膜合成、内容物和循环的机制， 和SVS的酶活性。此外，生产的 毛细胞特有的SV膜蛋白的免疫探针需要 孤立性SVS。青蛙球囊提供了一个很好的初步模型 发展中的微分离和纯化技术 体型相对较大。然而，在这方面有待完善的技术 试验项目，以及所获得的关于 SVS，应适用于哺乳动物模型。在这项初步研究中，我们 建议通过以下方法从蛙球囊毛细胞中分离出富含SV的部分 用酶法分离细胞，然后用细胞分级技术。我们 将通过以下方式证明分离部分中的囊泡有所丰富 一个可能的膜蛋白标志物的电子显微镜和免疫分析， 突触素。我们还将表征大豆的总蛋白质含量 富含SV组分的一维SDS-PAGE电泳法。建议数 项目需要使用微分离技术，因为 所涉及的组织数量极少。