Pulification and identification of substrate proteins for selective ubquitin-dependent proteolysis during NGF-induced neurite outgrowth of PC12h cells.

NGF 诱导 PC12h 细胞神经突生长期间选择性泛素依赖性蛋白水解的底物蛋白的纯化和鉴定。

基本信息

批准号：
10680723
负责人：
TAKADA Koji
金额：
$ 2.18万
依托单位：
JIKEI UNIVERSITY SCHOOL OF MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 1999
项目状态：
已结题

项目摘要

Optimization of purification protocols.Using K562 cells we established methods for purifying cellular ubiquitinated proteins. The first step is an immunoaffinity chromatography using a monoclonal antibody FK2 to ubiquitin. One ml of FK2-Sepharose gel was able to adsorb abount 0.2 mg of ubiquitin equivalent, and the adsorbed materials were completely eluted with 3..5 M MgClィイD22ィエD2. Then the materials were fractionated by gel filtration using Superdex 75, and two characteristic fractions having high molecular weights (HMW) (>30 kDa) and low molecular weights (LMW) (<30 kDa) were obtained. The former was mainly composed of multi-ubiquitinated proteins. In the latter we found ubiquitin thioesters with ubiquitin-conjugating enzymes (E2) as well as ubiquitinated proteins.Ubiquitinated protein variety during neurite elongation.By the combination of immunoprecipitation using FK2 antibody and SDS-PAGE, we visualized ubiquitinated proteins in PC12h cells cultivated in the presence of nerve gro … More wth factor (NGF). Three bands, 18, 19, and 36-kDa, newly appeared, and intensities of six bands, 20, 23, 26, 51, 68, and 93-kDa, increased.Purification of ubiquitinated proteins.Ubiquitinated protein mixtures (0.56 mg protein) were obtained from NGF-treated PC12h cells (2 x 10ィイD19ィエD1 cells) by the immunoaffinity chromatography. The mixtures were further separated by the gel filtration, and resulted in the two distinctive fractions of HMW and LMW. Several protein bands were isolated from the latter by SDS-PAGE, and amino acid sequencing of their peptide fragments obtained by lysyl-endopeptidase is ongoing in turns. The analysis of the 26-kDa band resulted in two amino acid sequences of ubiquitin digests and five unknown sequences.A monoclonal antibody against ubiquitin C-terminal tag peptide.This antibody is required for identification of the target proteins. Because of antigen insolubility, we had to change the peptide design, and finally established the desired antibody, which will be applied to the analysis of HMW ubiquitinated proteins. Less

纯化方案的优化。使用K562细胞，我们建立了纯化细胞泛素化蛋白的方法。第一步是使用单克隆抗体FK2的免疫亲属色谱法。一毫升的FK2-Sepharose凝胶能够吸附0.2 mg的泛素等效剂，并用3..5 m mgclii d22完全洗脱了吸附的材料。然后使用SuperDex 75通过凝胶过滤分割材料，并获得两个具有高分子量（HMW）（> 30 kDa）和低分子量（LMW）（LMW）（<30 kDa）的特征分数。前者主要由多泛素化蛋白组成。在后者中，我们发现了泛素二硫代蛋白（E2）以及泛素化的蛋白质。神经延长过程中启动蛋白质的变化。通过使用FK2抗体和SDS-PAGE进行免疫致敬的结合，我们可以看到ubiquicized uniquize prote prodimized uniquize uniquize uniquized uniquized uniquize unbiquize prof inbuquitized uniquized uniquized uniquize unbiquize pci12的细胞。（NGF）。新出现的三个乐队，18、19和36 kDa，六个乐队的强度，20、23、26、51、68和93 kDa，增加了。通过免疫亲和力色谱法获得了从NGF处理的PC12H细胞（2 x 10 d19 d1细胞）获得泛素化蛋白的纯化。混合物通过凝胶过滤进一步分离，并导致HMW和LMW的两个不同部分。通过SDS-PAGE从后者分离出几种蛋白质带，并持续进行通过赖氨基 - 内肽酶获得的肽片段的氨基酸测序。对26 kDa条带的分析导致了两个泛素蛋白消化的氨基酸序列和五个未知序列。一种针对泛素C-末端TAG肽的单克隆抗体。该抗体是鉴定靶蛋白所必需的。由于抗原的不可溶性，我们必须改变肽设计，最后建立了所需的抗体，该抗体将应用于HMW泛素化蛋白的分析。较少的