Getzoff T P, Zhu G, Bohnert H J, Jensen R G
Department of Molecular and Cellular Biology, University of Arizona, Tucson 85721-0088, USA.
Plant Physiol. 1998 Feb;116(2):695-702. doi: 10.1104/pp.116.2.695.
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.
编码1,5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）小亚基蛋白（S）的豌豆（Pisum sativum L.）RbcS 3A的cDNA，在花椰菜花叶病毒35S启动子的控制下已在拟南芥中表达，并检测到了转录本和成熟的S蛋白。特异性抗体在四种拟南芥S蛋白中显示出两个蛋白斑点，在豌豆S蛋白中显示出另外一个斑点。通过对部分纯化的酶制剂进行二维等电聚焦/十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离以及对银染蛋白斑点进行定量分析判断，嵌合Rubisco中的豌豆S蛋白占所有S蛋白的15%至18%。与野生型拟南芥Rubisco相比，嵌合酶的氨甲酰化位点少11±1%，羧化酶活性低11±1%。虽然豌豆S蛋白的表达、前体蛋白转运、加工和组装产生了一种稳定的全酶，但嵌合酶在催化方面的效率可重复性地较低。我们认为，每个全酶平均存在一个外源S蛋白是活性改变的原因。此外，与蓝细菌酶不同，高等植物Rubisco似乎已经进化出S蛋白与大亚基蛋白之间的物种特异性相互作用，这些相互作用参与活性位点的氨甲酰化。
