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.

使用胰蛋白酶和金黄色葡萄球菌 V8 蛋白酶对来自菠菜的 120 kDa 硝酸还原酶进行有限蛋白水解，获得蛋白水解片段。在 9 至 14 个 Edman 降解步骤中确定 NH4 末端序列，可以将片段精确定位在 CDNA 克隆 pSPNR117 的氨基酸序列中。该克隆插入片段长度为2324个碱基，其开放阅读框推导的氨基酸序列包含640个残基。与菠菜和菠菜的cDNA序列推导的氨基酸序列的序列同一性为61.2-80.1%。其他高等植物硝酸还原酶。然而，硝酸还原酶蛋白水解酶切位点周围的氨基酸序列同源性较差。通过 PH 5 处理、SE53、Con A-Sepharose 和层析，从菠菜叶中纯化了硝酸还原酶失活蛋白 16, 000 倍。色谱聚焦。 -收率为12%，比活性为115 u/mg。最终纯化的灭活剂的聚丙烯酰胺凝胶电泳产生 2 个主要蛋白质带，并且两条带均表现出硝酸还原酶灭活剂活性。通过凝胶过滤和 SDS 凝胶电泳分析该灭活剂蛋白，结果显示蛋白质可染色材料仅在分子量范围 110、000115、000 内。还原条件下的 SDS 凝胶电泳产生了 2 条对应于分子量 51、000 和 53 的蛋白条带, 00'0- 两个分离的亚基的蛋白水解图谱看起来相似并且可能相同。