Law R D, Plaxton W C
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Biochem J. 1995 May 1;307 ( Pt 3)(Pt 3):807-16. doi: 10.1042/bj3070807.
Phosphoenolpyruvate carboxylase (PEPC) from ripened banana (Musa cavendishii L.) fruits has been purified 127-fold to apparent homogeneity and a final specific activity of 32 mumol of oxaloacetate produced/min per mg of protein. Non-denaturing PAGE of the final preparation resolved a single protein-staining band that co-migrated with PEPC activity. Polypeptides of 103 (alpha-subunit) and 100 (beta-subunit) kDa, which stain for protein with equal intensity and cross-react strongly with anti-(maize leaf PEPC) immune serum, were observed following SDS/PAGE of the final preparation. CNBr cleavage patterns of the two subunits were similar, but not identical, suggesting that these polypeptides are related, but distinct, proteins. The enzyme's native molecular mass was estimated to be about 425 kDa. These data indicate that in contrast to the homotetrameric PEPC from most other sources, the banana fruit enzyme exists as an alpha 2 beta 2 heterotetramer. Monospecific rabbit anti-(banana PEPC) immune serum effectively immunoprecipitated the activity of the purified enzyme. Immunoblotting studies established that the 100 kDa subunit did not arise via proteolysis of the 103 kDa subunit after tissue extraction, and that the subunit composition of banana PEPC remains uniform throughout the ripening process. PEPC displayed a typical pH activity profile with an alkaline optimum and activity rapidly decreasing below pH 7.0. Enzymic activity was absolutely dependent on the presence of a bivalent metal cation, with Mg2+ or Mn2+ fulfilling this requirement. The response of the PEPC activity to PEP concentration and to various effectors was greatly influenced by pH and glycerol addition to the assay. The enzyme was activated by hexose-monophosphates and potently inhibited by malate, succinate, aspartate and glutamate at pH 7.0, whereas the effect of these metabolites was considerably diminished or completely abolished at pH 8.0. The significance of metabolite regulation of PEPC is discussed in relation to possible functions of this enzyme in banana fruit metabolism.
成熟香蕉（Musa cavendishii L.）果实中的磷酸烯醇式丙酮酸羧化酶（PEPC）已被纯化127倍，达到表观均一性，最终比活性为每毫克蛋白质每分钟产生32 μmol草酰乙酸。最终制剂的非变性聚丙烯酰胺凝胶电泳（PAGE）分离出一条与PEPC活性共迁移的单一蛋白质染色带。最终制剂经十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）后，观察到103 kDa（α亚基）和100 kDa（β亚基）的多肽，它们与蛋白质的染色强度相同，并与抗（玉米叶片PEPC）免疫血清发生强烈交叉反应。两个亚基的溴化氰裂解模式相似，但不完全相同，表明这些多肽是相关但不同的蛋白质。该酶的天然分子量估计约为425 kDa。这些数据表明，与大多数其他来源的同四聚体PEPC不同，香蕉果实中的酶以α2β2异四聚体形式存在。单特异性兔抗（香蕉PEPC）免疫血清有效地免疫沉淀了纯化酶的活性。免疫印迹研究表明，100 kDa亚基不是在组织提取后通过103 kDa亚基的蛋白水解产生的，并且香蕉PEPC的亚基组成在整个成熟过程中保持一致。PEPC呈现出典型的pH活性曲线，最适pH为碱性，pH低于7.0时活性迅速下降。酶活性绝对依赖于二价金属阳离子的存在，Mg2+或Mn2+满足这一要求。PEPC活性对磷酸烯醇式丙酮酸（PEP）浓度和各种效应物的反应受到pH和测定中甘油添加量的极大影响。该酶在pH 7.0时被己糖单磷酸激活，并被苹果酸、琥珀酸、天冬氨酸和谷氨酸强烈抑制，而在pH 8.0时这些代谢物的作用大大减弱或完全消除。结合该酶在香蕉果实代谢中可能的功能，讨论了PEPC代谢物调节的意义。
