Jiao J A, Chollet R
Department of Biochemistry, University of Nebraska-Lincoln 68583-0718.
Arch Biochem Biophys. 1989 Mar;269(2):526-35. doi: 10.1016/0003-9861(89)90136-7.
A reconstituted system composed of purified phosphoenolpyruvate carboxylase (PEP-Case) and a soluble protein kinase (PK) from green maize leaves was developed to critically assess the effects of in vitro protein phosphorylation on the catalytic and regulatory (malate sensitivity) properties of the target enzyme. The PK was partially purified from light-adapted leaf tissue by ammonium sulfate fractionation (0-60% saturation fraction) of a crude extract and blue dextran-agarose affinity chromatography. The resulting preparation was free of PEPCase. This partially purified protein kinase activated PEPCase from dark-adapted green maize leaves in an ATP-, Mg2+-, time-, and temperature-dependent fashion. Concomitant with these changes in PEPCase activity was a marked decrease in the target enzyme's sensitivity to feedback inhibition by L-malate. The PK-mediated incorporation of 32P from [gamma-32P]ATP into the protein substrate was directly correlated with these changes in PEPCase activity and malate sensitivity. The maximal molar 32P-incorporation value was about 0.25 per 100-kDa PEPCase subunit (i.e., 1 per holoenzyme). Phosphoamino acid analysis of the 32P-labeled target enzyme by two-dimensional thin-layer electrophoresis revealed the exclusive presence of phosphoserine. These in vitro results, together with our recent studies on the light-induced changes in phosphorylation status of green maize leaf PEPCase in vivo (J. A. Jiao and R. Chollet (1988) Arch. Biochem. Biophys. 261, 409-417), collectively provide the first unequivocal evidence that the seryl-phosphorylation of the dark-form enzyme by a soluble protein kinase is responsible for the changes in catalytic activity and malate sensitivity of C4 PEPCase observed in vivo during dark/light transitions of the parent leaf tissue.
构建了一个由纯化的磷酸烯醇式丙酮酸羧化酶（PEP羧化酶）和来自绿色玉米叶片的可溶性蛋白激酶（PK）组成的重组系统，以严格评估体外蛋白质磷酸化对靶酶催化和调节（苹果酸敏感性）特性的影响。通过对粗提物进行硫酸铵分级分离（0-60%饱和度级分）和蓝色葡聚糖-琼脂糖亲和色谱，从光适应的叶片组织中部分纯化了PK。所得制剂不含PEP羧化酶。这种部分纯化的蛋白激酶以ATP、Mg2+、时间和温度依赖性方式激活暗适应绿色玉米叶片中的PEP羧化酶。与PEP羧化酶活性的这些变化同时发生的是，靶酶对L-苹果酸反馈抑制的敏感性显著降低。PK介导的[γ-32P]ATP中32P掺入蛋白质底物与PEP羧化酶活性和苹果酸敏感性的这些变化直接相关。最大摩尔32P掺入值约为每100 kDa PEP羧化酶亚基0.25（即每全酶1个）。通过二维薄层电泳对32P标记的靶酶进行磷酸氨基酸分析，结果显示仅存在磷酸丝氨酸。这些体外结果，连同我们最近关于绿色玉米叶片PEP羧化酶在体内光诱导的磷酸化状态变化的研究（J. A. Jiao和R. Chollet（1988年）《生物化学与生物物理学报》261，409-417），共同提供了首个明确证据，即可溶性蛋白激酶对暗形式酶的丝氨酸磷酸化导致了在亲本叶片组织暗/光转变期间体内观察到的C4 PEP羧化酶催化活性和苹果酸敏感性的变化。
