权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

STUDIES ON INTRACELLULAR REGULATORY FACTORS INVOLVED IN AUTOPHAGIC PROTEOLYSIS

自噬蛋白水解相关细胞内调控因子的研究

基本信息

批准号：
09460061
负责人：
KADOWAKI Motoni
金额：
$ 8.64万
依托单位：
NIIGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

This research project deals with searches for unknown intracellular molecules involved in the regulation of autophagy, which contributes to bulk protein degradation in the cell. Results obtained through the project for these 2 years are as follows :1. Search for possible messenger molecules involved in amino acid signaling : Because of the importance of the quality of S.aureus alpha-toxin as a permeabilizing agent for the bioassay system, the method for culturing the bacteria and purifying the toxin was improved. The low MW extract of the liver stimulated by regulatory amino acids exhibited an inhibitory effect on autophagic proteolysis in alpha-toxin permeabilized hepatocytes. The active fraction was stable with heat, and stable at acidic and neutral pH, but unstable at alkaline pH, showing a positive charge. The possiblity that it may act on the autophagosome formation step which is regarded as a target site of amino acid action was indicated.2. Identification of the protein phosphor … More ylated by glucagon : In order to know the regulatory mechanism by glucagon, isolated rat hepatocytes was stimulated by glucagon with ^<32>Pi. A 49 kDa protein phosphorylated by glucagon in vivo was discovered in the crude lysosomal fraction, It was confirmed that it was phosphorylated by cAMP through protein kinase A in vitro.3. Maturation step of autophagy and GTP binding protein(s) : The permeabilized hepatocytes by streptolysin 0 (SLO), an another pore-forming toxin, was employed to clarify the maturation step of autophagy. From inhibitor studies, the involvement of some GTP binding proteins and an NEM sensitive protein was indicated at the maturation step. During the project, troubles with the potency of SLO took place, making it difficult to continue the assay. Thus, as an alternative method, an membranefusion assay between autophagosome and lysosome, an in vitro system, is now tried to be established. A cytosolic enzyme, betaine-homocystein methyltransferase, of which limited proteolytic intermediates after autophagic fusion can be identified immunochemically, is expected to make use as a probe for the fusion step. Less

该研究项目涉及寻找参与自噬调节的未知细胞内分子，自噬有助于细胞中大量蛋白质的降解。通过这两年的项目取得的成果如下：1.寻找参与氨基酸信号传导的可能信使分子：由于金黄色葡萄球菌α-毒素作为生物测定系统的透化剂的质量的重要性，改进了培养细菌和纯化毒素的方法。调节性氨基酸刺激的肝脏低分子量提取物对α-毒素透化肝细胞中的自噬蛋白水解具有抑制作用。活性部分对热稳定，并且在酸性和中性pH下稳定，但在碱性pH下不稳定，显示正电荷。提示其可能作用于自噬体的形成步骤，并将其作为氨基酸作用的靶位点.蛋白质磷的鉴定 ...更多信息胰高血糖素酰化：为了了解胰高血糖素的调节机制，用胰高血糖素和β Pi刺激分离的大鼠肝细胞<32>。在粗溶酶体组分中发现了胰高血糖素在体内磷酸化的49 kDa蛋白质，证实其在体外通过蛋白激酶A被cAMP磷酸化.自噬的成熟步骤和GTP结合蛋白：用另一种成孔毒素链球菌溶血素0（SLO）透化肝细胞，以阐明自噬的成熟步骤。从抑制剂的研究中，一些GTP结合蛋白和NEM敏感蛋白的参与表明在成熟步骤。在项目期间，SLO效价出现问题，难以继续进行测定。因此，作为一种替代方法，自噬体和溶酶体之间的膜融合试验，一个体外系统，现在试图建立。细胞质酶甜菜碱-高半胱氨酸甲基转移酶可以通过免疫化学方法鉴定自噬融合后有限的蛋白水解中间体，预计将用作融合步骤的探针。少