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Immuno-electron microscopy on the morphological marker enzymes and proteins for subcompartments of cell organellae.

免疫电子显微镜观察细胞器亚区室的形态标记酶和蛋白质。

基本信息

批准号：
05670005
负责人：
USUDA Nobuteru
金额：
$ 1.41万
依托单位：
Shinshu University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05670005/
关键词：
cell organellae marker enzymes immuno-electron microscopy paroxysm mitochondria nucleus

项目摘要

Morphological marker enzymes and proteins of subcompartments in cell organellae have been investigated, from two view points of the ultrastructure and protein molecules by immuno-electron microscopy.1.New findings were obtained mainly on the paroxysm, the mitochoindria and the nucleus.(1) Urate oxidase, which has been considered to be a common constituent of nucleoid core of mammalian hepatic peroxisomes, was demonstrated to be a morphological marker enzyme of vertebrate hepatic peroxisomes.(2) The process and functional aspect of the finding (1) was further investigated using cultured cells as materials.(3) Fatty acid beta-oxidation enzymes have been show to be commonly localized in the mitochondrial cristae, and suggested to be its marker.(4) Allantoinase was show to be a mitochondrial enzyme and as demonstrated to be a morphological marker enzyme of mitochondrial matrix.(5) A calcineurin isoform, CAP 50 protein and PCNA/cyclin were shown to be marker proteins for the nucleus.2.Problems in the research. Unexpectedly long time was needed to purify enzymes and proteins of endoplasmic reticulum, Golgi apparatus, and lysosomes. Even when they were purified, unexpected non-specific reaction existed in the antibodies. These two problem resulted in unsatisfactory results in these three cell organellas.3.Problem solutions. In order to achieve this object, immunoelectron microscopic studies employing the antibodies for synthetic polypeptides is being started.

本文从超微结构和蛋白质分子两个角度，用免疫电镜技术对细胞器亚区室的形态标志酶和蛋白质进行了研究：1.主要在阵发性、线粒体和细胞核上有新的发现。(1)尿酸氧化酶被认为是哺乳动物肝过氧化物酶体类核核心的共同组成部分，是脊椎动物肝过氧化物酶体的形态学标志酶。(2)以培养细胞为材料，进一步研究了发现（1）的过程和功能方面。(3)脂肪酸β-氧化酶通常位于线粒体嵴上，并被认为是其标志物。(4)尿囊素酶是一种线粒体酶，是线粒体基质的形态学标志酶。(5)CAP 50蛋白和PCNA/cyclin蛋白是细胞核的标志蛋白。2.研究中存在的问题。纯化内质网、高尔基体和溶酶体的酶和蛋白质需要出乎意料的长时间。即使纯化后，抗体中仍存在意想不到的非特异性反应。这两个问题导致在这三种细胞器中的结果不令人满意。为了实现这一目的，正在开始使用合成多肽的抗体进行免疫电子显微镜研究。