Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation
Chimeric Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase/oxygenase containing a pea small subunit protein is compromised in carbamylation
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DOI：
10.1104/pp.116.2.695
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发表时间：
1998-02-01
期刊：
影响因子：
7.4
通讯作者：
Jensen, RG
中科院分区：
文献类型：
--
作者：
Getzoff, TP;Zhu, GH;Jensen, RG
豌豆（Pisum sativum L.）RbcS 3A编码1，5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）的一个小亚基蛋白（S），在花椰菜花叶病毒35 S启动子的调控下，在拟南芥中得到了表达，并检测了其转录本和成熟S蛋白。特异性抗体揭示了四个拟南芥S的两个蛋白质点和豌豆S的一个额外的点。豌豆S嵌合Rubisco的15至18%的所有S，判断分离的二维等电聚焦/十二烷基硫酸钠-聚丙烯酰胺凝胶电泳凝胶从部分纯化的酶制剂和定量银染蛋白斑点。与野生型拟南芥Rubisco相比，嵌合酶的氨甲酰化位点减少了11 +/-1%，羧化酶活性降低了11 +/-1%。而豌豆S的表达，前蛋白运输，加工和组装导致在一个稳定的全酶，嵌合酶是可重复的催化效率较低。我们认为，平均而言，每个全酶一个外来S的存在是负责改变活动。此外，高等植物的Rubisco，不像蓝藻的酶，似乎已经发展了物种特异性的相互作用之间的S和大亚基蛋白，参与氨甲酰化的活性位点。
A cDNA of pea (Pisum sativum L.) RbcS 3A, encoding a small subunit protein (S) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), has been expressed in Arabidopsis thaliana under control of the cauliflower mosaic virus 35S promoter, and the transcript and mature S protein were detected. Specific antibodies revealed two protein spots for the four Arabidopsis S and one additional spot for pea S. Pea S in chimeric Rubisco amounted to 15 to 18% of all S, as judged by separation on two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels from partially purified enzyme preparations and quantitation of silver-stained protein spots. The chimeric enzyme had 11 +/- 1% fewer carbamylated sites and a 11 +/- 1% lower carboxylase activity than wild-type Arabidopsis Rubisco. Whereas pea S expression, preprotein transport, and processing and assembly resulted in a stable holoenzyme, the chimeric enzyme was reproducibly catalytically less efficient. We suggest that the presence of, on average, one foreign S per holoenzyme is responsible for the altered activity. In addition, higher-plant Rubisco, unlike the cyanobacterial enzyme, seems to have evolved species-specific interactions between S and the large subunit protein that are involved in carbamylation of the active site.