U.S. Department of Agriculture, Agricultural Research Service, Arid-Land Agricultural Research Center, 21881 N Cardon Lane, Maricopa, AZ 85138, USA.
Photosynth Res. 2011 Sep;108(2-3):143-55. doi: 10.1007/s11120-011-9667-8. Epub 2011 Jul 5.
Rubisco frequently undergoes unproductive interactions with its sugar-phosphate substrate that stabilize active sites in an inactive conformation. Restoring catalytic competence to these sites requires the "molecular chiropractic" activity of Rubisco activase (activase). To make the study of activase more routine and physiologically relevant, an assay was devised for measuring activase activity in leaf extracts based on the ATP-dependent activation of inactive Rubisco. Control experiments with an Arabidopsis activase-deficient mutant confirmed that the rate of Rubisco activation was dependent on the concentration of activase in the extracts. Activase catalyzed Rubisco activation at rates equivalent to 9-14% catalytic sites per min in desalted extracts of Arabidopsis, camelina, tobacco, cotton, and wheat. Faster rates were observed in a transgenic line of Arabidopsis that expresses only the β-isoform of activase, whereas no activity was detected in a line that expresses only the α-isoform. Activase activity was also low or undetectable in rice, maize, and Chlamydomonas, revealing differences in the stability of the enzyme in different species. These differences are discussed in terms of the ability of activase subunits to remain associated or to reassociate into active oligomers when the stromal milieu is diluted by extraction. Finally, the temperature response of activase activity in leaf extracts differed for Arabidopsis, camelina, tobacco, and cotton, corresponding to the respective temperature responses of photosynthesis for each species. These results confirmed the exceptional thermal lability of activase at physiological ratios of activase to Rubisco.
Rubisco 经常与糖磷酸底物发生非生产性相互作用，这些相互作用稳定了活性部位的非活性构象。要使这些活性部位恢复催化能力，需要 Rubisco 激活酶（激活酶）的“分子整脊”活性。为了使激活酶的研究更加常规和生理相关，设计了一种基于 ATP 依赖性激活失活 Rubisco 的叶提取物中测量激活酶活性的测定法。用拟南芥激活酶缺陷突变体进行的对照实验证实，Rubisco 的激活速率取决于提取物中激活酶的浓度。激活酶以相当于每分钟每 min 催化部位 9-14%的速率催化 Rubisco 的激活，在表达激活酶仅β-同工型的拟南芥转基因系中观察到更快的速率，而在仅表达α-同工型的系中则未检测到活性。在水稻、玉米和衣藻中，激活酶活性也较低或无法检测到，这表明不同物种中酶的稳定性存在差异。这些差异是根据激活酶亚基在基质环境因提取而稀释时保持结合或重新组装成活性寡聚体的能力来讨论的。最后，叶提取物中激活酶活性的温度响应在拟南芥、荠蓝、烟草和棉花之间存在差异，这与每种物种光合作用的相应温度响应相对应。这些结果证实了在生理比例的激活酶与 Rubisco 下，激活酶具有异常的热不稳定性。
