Ceccarelli E A, Krapp A R, Serra E C, Carrillo N
Molecular Biology Division, PROMUBIE, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Eur J Biochem. 1996 May 15;238(1):192-7. doi: 10.1111/j.1432-1033.1996.0192q.x.
The cytosolic precursor of the chloroplast flavoprotein ferredoxin-NADP+ reductase was expressed in Escherichia coli rendering a soluble protein that contained bound FAD and could be imported by isolated chloroplasts. The mechanism of plastid translocation was studied under defined conditions using this recombinant precursor holoprotein and intact pea chloroplasts. The first step in the import pathway, namely, binding of the reductase precursor to isolated chloroplasts, was saturable at about 2000 molecules/plastid, and showed a high-affinity interaction with a dissociation constant Kd of approximately 5 nM. Binding was not affected by the addition of soluble leaf extracts or by prior denaturation of the precursor with urea. Analysis of the initial import rates at different precursor concentrations indicated the existence of a single translocation system for this protein. Inclusion of leaf extracts in the assay resulted in a three-fold increase of the maximal import rates to 14,000 molecules . min-(1).chloroplast-(1), with a concomitant decrease in the apparent Km for the recombinant precursor, from 1 microM to 100-150 nM. Comparison of Km and Kd values under various conditions indicated that the binding step of the translocation process is largely irreversible, favouring import and processing. In the absence of extract, a denatured precursor obtained by incubation with urea was a better substrate for plastid import than the holoprotein. Treatment of the precursor with either extract or urea resulted in similar increases in import efficiency (V/Km), suggesting that stimulation by leaf extracts is probably related to unfolding of the precursor prior to translocation.
叶绿体黄素蛋白铁氧化还原蛋白-NADP⁺还原酶的胞质前体在大肠杆菌中表达，产生一种可溶性蛋白，该蛋白含有结合的黄素腺嘌呤二核苷酸（FAD），并且可以被分离的叶绿体导入。使用这种重组前体全蛋白和完整的豌豆叶绿体，在特定条件下研究了质体转运机制。导入途径的第一步，即还原酶前体与分离的叶绿体的结合，在约2000个分子/质体时达到饱和，并显示出与解离常数Kd约为5 nM的高亲和力相互作用。添加可溶性叶提取物或用尿素对前体进行预变性均不影响结合。对不同前体浓度下的初始导入速率进行分析表明，该蛋白存在单一的转运系统。在测定中加入叶提取物导致最大导入速率增加了三倍，达到14,000个分子·分钟⁻¹·质体⁻¹，同时重组前体的表观米氏常数（Km）从1 μM降至100 - 150 nM。在各种条件下比较Km和Kd值表明，转运过程的结合步骤在很大程度上是不可逆的，有利于导入和加工。在没有提取物的情况下，通过与尿素孵育获得的变性前体比全蛋白更适合质体导入。用提取物或尿素处理前体导致导入效率（V/Km）有类似的增加，这表明叶提取物的刺激可能与前体在转运之前的解折叠有关。
