Kosar-Hashemi Behjat, Irwin Jennifer A, Higgins Jody, Rahman Sadequr, Morell Matthew K
Commonwealth Scientific and Industrial Research Organisation Plant Industry, Canberra, ACT, Australia.
Electrophoresis. 2006 May;27(9):1832-9. doi: 10.1002/elps.200500400.
A 2-D affinity electrophoretic technique (2-DAE) has been used to isolate proteins that interact with various starch components from total barley endosperm extracts. In the first dimension, proteins are separated by native PAGE. The second-dimensional gel contains polysaccharides such as amylopectin and glycogen. The migration of starch-interacting proteins in this dimension is determined by their affinity towards a particular polysaccharide and these proteins are therefore spatially separated from the bulk of proteins in the crude extract. Four distinct proteins demonstrate significant affinity for amylopectin and have been identified as starch branching enzyme I (SBEI), starch branching enzyme IIa (SBEIIa), SBEIIb and starch phosphorylase using polyclonal antibodies and zymogram activity analysis. In the case of starch phosphorylase, a protein spot was excised from a 2-DAE polyacrylamide gel and analysed using Q-TOF MS/MS, resulting in the alignment of three internal peptide sequences with the known sequence of the wheat plastidic starch phosphorylase isoform. This assignment was confirmed by the determination of the enzyme's function using zymogram analysis. Dissociation constants (Kd) were calculated for the three enzymes at 4 degrees C and values of 0.20, 0.21 and 1.3 g/L were determined for SBEI, SBEIIa and starch phosphorylase, respectively. Starch synthase I could also be resolved from the other proteins in the presence of glycogen and its identity was confirmed using a polyclonal antibody and by activity analysis. The 2-DAE method described here is simple, though powerful, enabling protein separation from crude extracts on the basis of function.
一种二维亲和电泳技术（2-DAE）已被用于从大麦胚乳总提取物中分离与各种淀粉成分相互作用的蛋白质。在第一维中，蛋白质通过非变性聚丙烯酰胺凝胶电泳（Native PAGE）进行分离。第二维凝胶含有支链淀粉和糖原等多糖。淀粉相互作用蛋白在这一维中的迁移取决于它们对特定多糖的亲和力，因此这些蛋白在空间上与粗提物中的大部分蛋白分离。使用多克隆抗体和酶谱活性分析，四种不同的蛋白质对支链淀粉表现出显著的亲和力，并已被鉴定为淀粉分支酶I（SBEI）、淀粉分支酶IIa（SBEIIa）、SBEIIb和淀粉磷酸化酶。对于淀粉磷酸化酶，从2-DAE聚丙烯酰胺凝胶中切下一个蛋白点，并用Q-TOF MS/MS进行分析，结果得到三个内部肽序列与小麦质体淀粉磷酸化酶同工型的已知序列比对。通过酶谱分析确定该酶的功能，证实了这一鉴定。在4℃下计算了这三种酶的解离常数（Kd），SBEI、SBEIIa和淀粉磷酸化酶的值分别为0.20、0.21和1.3 g/L。在糖原存在的情况下，淀粉合酶I也可以从其他蛋白质中分离出来，并用多克隆抗体和活性分析证实了其身份。这里描述的2-DAE方法虽然简单，但功能强大，能够基于功能从粗提物中分离蛋白质。
