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

Intracellular fate of nitrate reductase in higher plants

高等植物硝酸还原酶的细胞内命运

基本信息

批准号：
01540558
负责人：
NAKAGAWA Hiroki
金额：
$ 1.22万
依托单位：
Chiba University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1991
项目状态：
已结题

项目摘要

Proteolytic fragments were obtained by limited proteolysis of 120 kDa nitrate reductase from Spinacia oleracea L. using trypsin and Staphylococcus aureus V8 protease. Determination of NH4-terminal sequences in 9 to 14 Edman degradation steps allowed the exact localization of the fragments within the amino-acid sequence of CDNA clone pSPNR117. This clone has a 2324 base insert, and the amino acid sequence deduced from its open reading frame, which contains 640 residues.A sequence identity of 61.2-80.1% was found in the amino acid sequences deduced from the CDNA sequences as obtained by spinach and other higher plant nitrate reductases. However, the amino acid sequences surrounding the proteolytic cleavage sites of nitrate reductase had poor homology.A nitrate reductase-inactivator protein has been purified 16, 000-fold from spinach leaves by PH 5 treatment, chromatography on SE53, Con A-Sepharose, and chromatofocusing. -The yield was 12%, the specific activity was 115 u/mg. Polyacrylamide gel electrophoresis of the final purified inactivator yield 2 major protein bands and both bands exhibited nitrate reductase-inactivator activity. Analysis of this inactivator protein by gel filtration and SDS-gel electrophoresis revealed protein stainable material only in a molecular weight range of 110, 000115, 000. SDS gel electrophoresis under reducing conditions yielded 2 protein bands corresponding to molecular weight of 51, 000 and 53, 00'0- The proteolytic mapping for the two separated subunits appeared similar and possibly identical.

用胰酶和金黄色葡萄球菌V8酶对菠菜120 kDa硝酸还原酶进行限制性酶解，得到蛋白水解酶片段。通过测定9到14个Edman降解步骤中的NH4末端序列，可以准确地定位CDNA克隆pSPNR117的氨基酸序列中的片段。该克隆有一个2324碱基的插入片段，由其开放阅读框推导出的氨基酸序列包含640个残基，与菠菜等高等植物硝酸还原酶序列推导的氨基酸序列同源性为61.2-80.1%。经过PH5处理、SE53柱层析、ConA-Sepharose柱层析和层析等步骤，从菠菜叶片中分离纯化出一种硝酸还原酶失活蛋白，纯化倍数为16000倍。-产率为12%，比活性为115u/mg。最终纯化的灭活剂经聚丙烯酰胺凝胶电泳法得到2条主要蛋白条带，两条带均具有硝酸还原酶灭活活性。通过凝胶过滤和十二烷基硫酸钠-凝胶电泳法对该灭活蛋白进行分析，发现蛋白染色物质仅在110000,000115,000的分子量范围内。在还原条件下的十二烷基硫酸钠凝胶电泳法得到了两条蛋白质带，其相对分子质量分别为51,000和53，00‘0，两个分离的亚基的蛋白质降解图谱相似，甚至可能相同。