López Jaramillo J, Chueca A, Jacquot J P, Hermoso R, Lázaro J J, Sahrawy M, López Gorgé J
Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Plant Physiol. 1997 Aug;114(4):1169-75. doi: 10.1104/pp.114.4.1169.
A cDNA clone encoding pea (Pisum sativum L.) chloroplast thioredoxin (Trx) m and its transit peptide were isolated from a pea cDNA library. Its deduced amino acid sequence showed 70% homology with spinach (Spinacia oleracea L.) Trx m and 25% homology with Trx f from pea and spinach. After subcloning in the Ndel-BamHI sites of pET-12a, the recombinant supplied 20 mg Trx m/L. Escherichia coli culture. This protein had 108 amino acids and was 12,000 D, which is identical to the pea leaf native protein. Unlike pea Trx f, pea Trx m showed a hyperbolic saturation of pea chloroplast fructose-1,6-bisphosphatase (FBPase), with a Trx m/ FBPase molar saturation ratio of about 60, compared with 4 for the Trx f/FBPase quotient. Cross-experiments have shown the ability of pea Trx m to activate the spinach chloroplast FBPase, results that are in contrast with those in spinach found by P. Schürmann, K. Maeda, and A. Tsugita ([1981] Eur J Biochem 116: 37-45), who did not find Trx m efficiency in FBPase activation. This higher efficiency of pea Trx m could be related to the presence of four basic residues (arginine-37, lysine-70, arginine-74, and lysine-97) flanking the regulatory cluster; spinach Trx m lacks the positive charge corresponding to lysine-70 of pea Trx m. This has been confirmed by K70E mutagenesis of pea Trx m, which leads to a 50% decrease in FBPase activation.
从豌豆cDNA文库中分离出一个编码豌豆（Pisum sativum L.）叶绿体硫氧还蛋白（Trx）m及其转运肽的cDNA克隆。其推导的氨基酸序列与菠菜（Spinacia oleracea L.）Trx m具有70%的同源性，与豌豆和菠菜的Trx f具有25%的同源性。在亚克隆到pET-12a的Ndel-BamHI位点后，重组体在大肠杆菌培养物中提供了20mg Trx m/L。该蛋白有108个氨基酸，分子量为12000D，与豌豆叶片天然蛋白相同。与豌豆Trx f不同，豌豆Trx m对豌豆叶绿体果糖-1,6-二磷酸酶（FBPase）表现出双曲线饱和度，Trx m/FBPase摩尔饱和度比约为60，而Trx f/FBPase商为4。交叉实验表明豌豆Trx m能够激活菠菜叶绿体FBPase，这一结果与P. Schürmann、K. Maeda和A. Tsugita（[1981] Eur J Biochem 116: 37-45）在菠菜中发现的结果相反，他们没有发现Trx m在激活FBPase方面的效率。豌豆Trx m的这种更高效率可能与调节簇两侧存在四个碱性残基（精氨酸-37、赖氨酸-70、精氨酸-74和赖氨酸-97）有关；菠菜Trx m缺乏与豌豆Trx m赖氨酸-70相对应的正电荷。豌豆Trx m的K70E诱变证实了这一点，该诱变导致FBPase激活降低50%。
