Kleczkowski L A, Villand P, Preiss J, Olsen O A
Plant Molecular Biology Laboratory, NLVF, Aas, Norway.
J Biol Chem. 1993 Mar 25;268(9):6228-33.
Barley (Hordeum vulgare, cv. Bomi) leaf ADP-glucose pyrophosphorylase (AGP) was purified to near-homogeneity, using ammonium sulfate fractionation and heat treatment as well as ion exchange, hydrophobic, and dye-ligand chromatography. The enzyme was found to be composed of two subunit types of 51 and 54 kDa, which were recognized by the respective rabbit antibodies against the small and/or large subunit of spinach leaf AGP. Substrate kinetics and product inhibition studies, carried out in the direction of ADP-glucose synthesis, suggested a sequential Iso Ordered Bi Bi kinetic mechanism for the enzyme. In addition, inhibition patterns with CrATP, a dead-end inhibitor of ATP-utilizing enzymes, were consistent with ATP (synthesis reaction) and ADP-glucose (pyrophosphorolysis reaction) binding first to different free forms of the enzyme. The AGP was potently activated by 3-phosphoglycerate (PGA) (K alpha of 5 microM) and inhibited by orthophosphate (P(i)), with the ratio of the two effectors playing a major role in modulating AGP activity. At low [PGA]/[P(i)] ratios, P(i) could reverse the activating effect of PGA. However, at the [PGA]/[Pi] ratios of 1.4, or higher, some activating effect of P(i) in the presence of PGA was observed. PGA decreased the Km values for all substrates of AGP (in both the synthesis and pyrophosphorolysis directions). Properties of the barley leaf AGP are compared to those of AGP from barley seed endosperm, which is insensitive to PGA/P(i) regulation.
利用硫酸铵分级分离、热处理以及离子交换色谱、疏水色谱和染料配体色谱法，将大麦（Hordeum vulgare，品种Bomi）叶片中的ADP-葡萄糖焦磷酸化酶（AGP）纯化至接近均一。该酶由51 kDa和54 kDa两种亚基类型组成，分别被针对菠菜叶片AGP小亚基和/或大亚基的兔抗体识别。在ADP-葡萄糖合成方向上进行的底物动力学和产物抑制研究表明，该酶具有有序的双底物双产物顺序动力学机制。此外，ATP利用酶的终产物抑制剂CrATP的抑制模式与ATP（合成反应）和ADP-葡萄糖（焦磷酸解反应）首先结合到酶的不同游离形式一致。AGP被3-磷酸甘油酸（PGA）（Kα为5 μM）强烈激活，并被正磷酸盐（P(i)）抑制，两种效应物的比例在调节AGP活性中起主要作用。在低[PGA]/[P(i)]比例下，P(i)可逆转PGA的激活作用。然而，在[PGA]/[Pi]比例为1.4或更高时，在PGA存在下观察到P(i)有一些激活作用。PGA降低了AGP所有底物的Km值（在合成和焦磷酸解两个方向上）。将大麦叶片AGP的特性与大麦种子胚乳中的AGP特性进行了比较，后者对PGA/P(i)调节不敏感。
