Starch synthesis is an elaborate process employing several isoforms of starch synthases (SSs), starch branching enzymes (SBEs) and debranching enzymes (DBEs). In cereals, some starch biosynthetic enzymes can form heteromeric complexes whose assembly is controlled by protein phosphorylation. Previous studies suggested that SSIIa forms a trimeric complex with SBEIIb, SSI, in which SBEIIb is phosphorylated. This study investigates the post‐translational modification of SSIIa, and its interactions with SSI and SBEIIb in maize amyloplast stroma. SSIIa, immunopurified and shown to be free from other soluble starch synthases, was shown to be readily phosphorylated, affecting V_max but with minor effects on substrate K_d and K_m values, resulting in a 12‐fold increase in activity compared with the dephosphorylated enzyme. This ATP‐dependent stimulation of activity was associated with interaction with SBEIIb, suggesting that the availability of glucan branching limits SSIIa and is enhanced by physical interaction of the two enzymes. Immunoblotting of maize amyloplast extracts following non‐denaturing polyacrylamide gel electrophoresis identified multiple bands of SSIIa, the electrophoretic mobilities of which were markedly altered by conditions that affected protein phosphorylation, including protein kinase inhibitors. Separation of heteromeric enzyme complexes by GPC, following alteration of protein phosphorylation states, indicated that such complexes are stable and may partition into larger and smaller complexes. The results suggest a dual role for protein phosphorylation in promoting association and dissociation of SSIIa‐containing heteromeric enzyme complexes in the maize amyloplast stroma, providing new insights into the regulation of starch biosynthesis in plants.

中文翻译：

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蛋白质磷酸化调节玉米胚乳淀粉合酶IIa活性和蛋白质-蛋白质相互作用

淀粉合成是一个复杂的过程，它使用淀粉合酶（SS），淀粉分支酶（SBE）和脱支酶（DBE）的几种同工型。在谷物中，某些淀粉生物合成酶可以形成异聚复合物，其组装受蛋白质磷酸化控制。先前的研究表明，SSIIa与SBEIIb，SSI形成三聚体复合物，其中SBEIIb被磷酸化。这项研究调查了玉米淀粉质基质中SSIIa的翻译后修饰及其与SSI和SBEIIb的相互作用。经免疫纯化并显示不含其他可溶性淀粉合酶的SSIIa已显示易于磷酸化，影响V _max，但对底物K _d和K _m的影响较小与去磷酸化酶相比，其活性增加了12倍。这种ATP依赖性的活性刺激与与SBEIIb的相互作用有关，这表明葡聚糖分支的可利用性限制了SSIIa，并通过两种酶的物理相互作用而得到增强。通过非变性聚丙烯酰胺凝胶电泳对玉米淀粉体提取物进行免疫印迹，鉴定出多个SSIIa条带，其电泳迁移率受到影响蛋白磷酸化的条件（包括蛋白激酶抑制剂）的影响而明显改变。在蛋白质磷酸化状态改变后，通过GPC分离异聚酶复合物表明，这种复合物是稳定的，并且可能分成更大和更小的复合物。