Moraes T F, Plaxton W C
Departments of Biochemistry and Biology, Queen's University, Kingston, Ontario, Canada.
Eur J Biochem. 2000 Jul;267(14):4465-76. doi: 10.1046/j.1432-1327.2000.01495.x.
Phosphoenolpyruvate carboxylase (PEPC) specific activity increased by 250% following 8 to 10 days of Pi starvation of Brassica napus suspension cells. Densitometric scanning of PEPC immunoblots revealed a close correlation between PEPC activity and the amount of the antigenic 104-kDa PEPC subunit. To further assess the influence of Pi deprivation on PEPC, the enzyme was purified from Pi-sufficient (+Pi) and Pi-starved (-Pi) cells to electrophoretic homogeneity and final specific activities of 37-40 micromol phosphoenolpyruvate utilized per min per mg protein. Gel filtration, SDS/PAGE, and CNBr peptide mapping indicated that the +Pi and -Pi PEPCs are both homotetramers composed of an identical 104-kDa subunit. Respective pH-activity profiles, phosphoenolpyruvate saturation kinetics, and sensitivity to L-malate inhibition were also indistinguishable. Kinetic studies and phosphatase treatments revealed that PEPC of the +Pi and -Pi cells exists mainly in its dephosphorylated (L-malate sensitive) form. Thus, up-regulation of PEPC activity in -Pi cells appears to be solely due to the accumulation of the same PEPC isoform being expressed in +Pi cells. PEPC activity was modulated by several metabolites involved in carbon and nitrogen metabolism. At pH 7.3, marked activation by glucose 6-phosphate and inhibition by L-malate, L-aspartate, L-glutamate, DL-isocitrate, rutin and quercetin was observed. The following paper provides a model for the coordinate regulation of B. napus PEPC and cytosolic pyruvate kinase by allosteric effectors. L-Aspartate and L-glutamate appear to play a crucial role in the control of the phosphoenolpyruvate branchpoint in B. napus, particularly with respect to the integration of carbohydrate partitioning with the generation of carbon skeletons required during nitrogen assimilation.
在甘蓝型油菜悬浮细胞缺磷8至10天后，磷酸烯醇式丙酮酸羧化酶（PEPC）的比活性增加了250%。对PEPC免疫印迹进行光密度扫描显示，PEPC活性与104-kDa PEPC亚基抗原量之间存在密切相关性。为了进一步评估缺磷对PEPC的影响，从磷充足（+Pi）和缺磷（-Pi）的细胞中纯化该酶至电泳纯，最终比活性为每分钟每毫克蛋白质利用37 - 40微摩尔磷酸烯醇式丙酮酸。凝胶过滤、SDS/PAGE和CNBr肽图谱分析表明，+Pi和-Pi的PEPC均为由相同的104-kDa亚基组成的同四聚体。各自的pH-活性曲线、磷酸烯醇式丙酮酸饱和动力学以及对L-苹果酸抑制的敏感性也无差异。动力学研究和磷酸酶处理表明，+Pi和-Pi细胞中的PEPC主要以其去磷酸化（对L-苹果酸敏感）形式存在。因此，-Pi细胞中PEPC活性的上调似乎完全是由于在+Pi细胞中表达的相同PEPC同工型的积累。PEPC活性受到参与碳和氮代谢的几种代谢物的调节。在pH 7.3时，观察到6-磷酸葡萄糖有明显激活作用，而L-苹果酸、L-天冬氨酸、L-谷氨酸、DL-异柠檬酸、芦丁和槲皮素有抑制作用。以下论文提供了一个变构效应物对甘蓝型油菜PEPC和胞质丙酮酸激酶进行协同调节的模型。L-天冬氨酸和L-谷氨酸似乎在甘蓝型油菜磷酸烯醇式丙酮酸分支点的控制中起着关键作用，特别是在碳水化合物分配与氮同化过程中所需碳骨架生成的整合方面。
