Duff S M, Lefebvre D D, Plaxton W C
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Arch Biochem Biophys. 1991 Apr;286(1):226-32. doi: 10.1016/0003-9861(91)90033-f.
An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.
从黑芥（黑芥菜）叶叶柄细胞悬浮培养物中纯化出一种酸性磷酸酶，纯化倍数达1633倍，最终比活性为1225（每分钟产生的正磷酸盐微摩尔数/毫克蛋白质），且接近均一。天然蛋白质是一种糖基化单体，分子量为60 kDa，pI为4.5。该酶的最适pH范围较宽，约为pH 5.6，且热稳定。最终制剂能水解多种磷酸酯。对3-磷酸甘油酸、2,3-二磷酸甘油酸、焦磷酸（PPi）和磷酸烯醇式丙酮酸（PEP）的特异性常数最高。该酶被4 mM Mg2+或Mn2+激活1.4倍，但被钼、磷酸根离子、氟和几种磷酸化化合物强烈抑制。亚细胞定位实验表明，这种非特异性酸性磷酸酶可能是一种分泌酶，定位于细胞壁。相比之下，黑芥PEP磷酸酶似乎定位于细胞液泡中。通过CNBr裂解进行肽图谱分析，以研究这两种磷酸酶的结构相关性。它们各自的CNBr裂解模式不同，表明黑芥酸性磷酸酶和PEP磷酸酶是不同的多肽。结合黑芥细胞悬浮培养物对营养性磷酸盐缺乏的生化适应性，讨论了这两种磷酸酶可能的代谢功能。
