Gupta Sanjay K, Sowokinos Joseph R, Hahn In-Su
Department of Horticultural Science, University of Minnesota, 305 Alderman Hall, 1970 Folwell Avenue, St. Paul, MN 55108, USA.
J Plant Physiol. 2008 May 5;165(7):679-90. doi: 10.1016/j.jplph.2007.09.001. Epub 2007 Nov 8.
The regulation of UDP-Glc pyrophosphorylase (UGPase) isozyme, UGP5, was investigated in potato tuber. The cDNA for UGP5 was cloned into the bacterial expression vector pET21d and recombinant (RC) enzyme was expressed in E. coli (BL21 star cells). The RC-UGP5 isozyme was purified to near homogeneity using salt precipitation, hydrophobic interaction, and anion-exchange column chromatography. Kinetic analysis revealed that in the synthesis direction, K(m) values for Glc-1-P (0.83 mM) and UTP (0.22 mM) were similar to those observed previously with the mother tuber (MT)-UGP5. In the pyrophosphorolysis direction, the K(m) values for UDP-Glc (0.68 mM) and PPi (0.56 mM) were slightly higher than those observed previously. Maximum reaction velocities (V(max)) for RC-UGP5 were also elevated. Since the molecular mass, charge, and amino acid sequence of the MT- and RC-UGP5 isozymes were identical, it was assumed that altered kinetic constants may be due to an improper folding of RC-UGP5 polypeptide. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and proteomic analysis demonstrated that the UGP5 isozyme was a single polypeptide with a calculated molecular mass of 51.8kDa consisting of 477 amino acids. Native PAGE and kinetic analysis revealed that this polypeptide was monomeric in nature. Immunoblotting with specific antibodies and LC-MS/MS data indicated that UGP5 did not require any post-translational modification (e.g., phosphorylation, O-glycosylation, oligomerization/de-oligomerization, or the presence of the regulatory 14-3-3 proteins) for its regulation. Additionally, the two closely associated isozymes UGP5 and UGP6 in the cv. Snowden are likely the result of allelic differences of UGPase at a single locus.
在马铃薯块茎中研究了UDP - 葡萄糖焦磷酸化酶（UGPase）同工酶UGP5的调控。将UGP5的cDNA克隆到细菌表达载体pET21d中，并在大肠杆菌（BL21 star细胞）中表达重组（RC）酶。使用盐沉淀、疏水相互作用和阴离子交换柱色谱法将RC - UGP5同工酶纯化至接近均一。动力学分析表明，在合成方向上，Glc - 1 - P（0.83 mM）和UTP（0.22 mM）的K(m)值与先前在母块茎（MT） - UGP5中观察到的值相似。在焦磷酸解方向上，UDP - 葡萄糖（0.68 mM）和PPi（0.56 mM）的K(m)值略高于先前观察到的值。RC - UGP5的最大反应速度（V(max)）也有所提高。由于MT - 和RC - UGP5同工酶的分子量、电荷和氨基酸序列相同，推测动力学常数的改变可能是由于RC - UGP5多肽折叠不当所致。液相色谱 - 串联质谱（LC - MS/MS）和蛋白质组学分析表明，UGP5同工酶是一种单一多肽，计算分子量为51.8 kDa，由477个氨基酸组成。天然PAGE和动力学分析表明该多肽本质上是单体。用特异性抗体进行免疫印迹和LC - MS/MS数据表明，UGP5的调控不需要任何翻译后修饰（例如磷酸化、O - 糖基化、寡聚化/去寡聚化或存在调节性14 - 3 - 3蛋白）。此外，品种Snowden中两个紧密相关的同工酶UGP5和UGP6可能是UGPase在单个位点上等位基因差异的结果。
