Department of Plant Physiology and Biophysics, Institute of Molecular and Cell Biology, University of Tartu, Riia 23, Tartu 51010, Estonia.
J Exp Bot. 2009;60(14):4077-88. doi: 10.1093/jxb/erp242. Epub 2009 Aug 6.
Site turnover rate (k(cat)) of Rubisco was measured in intact leaves of different plants. Potato (Solanum tuberosum L.) and birch (Betula pendula Roth.) leaves were taken from field-growing plants. Sunflower (Helianthus annuus L.), wild type (wt), Rubisco-deficient (-RBC), FNR-deficient (-FNR), and Cyt b(6)f deficient (-CBF) transgenic tobacco (Nicotiana tabacum L.) were grown in a growth chamber. Rubisco protein was measured with quantitative SDS-PAGE and FNR protein content with quantitative immunoblotting. The Cyt b(6)f level was measured in planta by maximum electron transport rate and the photosystem I (PSI) content was assessed by titration with far-red light. The CO(2) response of Rubisco was measured in planta with a fast-response gas exchange system at maximum ribulose 1,5-bisphosphate concentration. Reaction site k(cat) was calculated from V(m) and Rubisco content. Biological variation of k(cat) was significant, ranging from 1.5 to 4 s(-1) in wt, but was >6 s(-1) at 23 degrees C in -RBC leaves. The lowest k(cat) of 0.5 s(-1) was measured in -FNR and -CBF plants containing sufficient Rubisco but having slow electron transport rates. Plotting k(cat) against PSI per Rubisco site resulted in a hyperbolic relationship where wt plants are on the initial slope. A model is suggested in which Rubisco Activase is converted into an active ATP-form on thylakoid membranes with the help of a factor related to electron transport. The activation of Rubisco is accompanied by the conversion of the ATP-form into an inactive ADP-form. The ATP and ADP forms of Activase shuttle between thylakoid membranes and stromally-located Rubisco. In normal wt plants the electron transport-related activation of Activase is rate-limiting, maintaining 50-70% Rubisco sites in the inactive state.
Rubisco 的比活（k(cat)）通过测定不同植物的完整叶片来衡量。马铃薯（Solanum tuberosum L.）和桦树（Betula pendula Roth.）叶片取自大田生长的植株。向日葵（Helianthus annuus L.）、野生型（wt）、Rubisco 缺乏型（-RBC）、FNR 缺乏型（-FNR）和 Cyt b(6)f 缺乏型（-CBF）烟草（Nicotiana tabacum L.）转基因植株在生长室中生长。Rubisco 蛋白通过定量 SDS-PAGE 测定，FNR 蛋白含量通过定量免疫印迹测定。Cyt b(6)f 水平通过最大电子传递速率进行体内测定，通过远红光滴定评估光系统 I（PSI）含量。Rubisco 的 CO(2)响应通过最大核酮糖 1,5-二磷酸浓度下快速响应气体交换系统进行体内测量。反应部位 k(cat)根据 V(m)和 Rubisco 含量计算。wt 中 k(cat)的生物变异性显著，范围为 1.5-4 s(-1)，但在 -RBC 叶片中，在 23 摄氏度时大于 6 s(-1)。在含有足够 Rubisco 但电子传递速率较慢的 -FNR 和 -CBF 植物中，测量到的最低 k(cat)为 0.5 s(-1)。将 k(cat)与 Rubisco 位点上的 PSI 作图，得到一条双曲线关系，wt 植物位于初始斜率上。提出了一个模型，其中 Rubisco Activase 在类囊体膜上借助与电子传递相关的因子转化为活性 ATP 形式。Rubisco 的激活伴随着 ATP 形式转化为无活性 ADP 形式。Activase 的 ATP 和 ADP 形式在类囊体膜和基质定位的 Rubisco 之间穿梭。在正常的 wt 植物中，与电子传递相关的 Activase 激活是限速步骤，将 50-70%的 Rubisco 位点保持在无活性状态。
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