Intracellular fate of nitrate reductase in higher plants

利用胰蛋白酶和金黄色葡萄球菌V8蛋白酶对菠菜甘蓝中120 kDa的硝酸还原酶进行有限的蛋白水解，获得蛋白水解片段。通过测定9 ~ 14个Edman降解步骤的nh4末端序列，可以准确定位CDNA克隆pSPNR117的氨基酸序列。该克隆有2324个碱基插入，从其开放阅读框推断出包含640个残基的氨基酸序列。由菠菜和其他高等植物硝酸还原酶的CDNA序列推导出的氨基酸序列，序列一致性为61.2 ~ 80.1%。然而，硝酸盐还原酶蛋白水解裂解位点周围的氨基酸序列同源性较差。通过PH 5处理、SE53层析、Con A- sepharose层析和色谱聚焦，从菠菜叶中纯化出一种硝酸还原酶失活蛋白，纯度达到16000倍。-产率为12%，比活性为115 u/mg。最终纯化的失活剂聚丙烯酰胺凝胶电泳得到2个主要的蛋白条带，两个条带都表现出硝酸还原酶-失活剂的活性。通过凝胶过滤和sds -凝胶电泳对该失活蛋白进行分析，发现该蛋白仅在分子量为11万~ 11.5万的范围内具有可染性。在还原条件下，SDS凝胶电泳得到了两条分子量分别为51,000和53,000 '0的蛋白条带-两个分离亚基的蛋白水解图谱相似，甚至可能相同。

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.