Cashikar A. G., Kumaresan R., Rao N. M.
Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.
Plant Physiol. 1997 Jul;114(3):907-915. doi: 10.1104/pp.114.3.907.
Phosphatases are known to play a crucial role in phosphate turnover in plants. However, the exact role of acid phosphatases in plants has been elusive because of insufficient knowledge of their in vivo substrate and subcellular localization. We investigated the biochemical properties of a purple acid phosphatase isolated from red kidney bean (Phaseolus vulgaris) (KBPAP) with respect to its substrate and inhibitor profiles. The kinetic parameters were estimated for five substrates. We used 31P nuclear magnetic resonance to investigate the in vivo substrate of KBPAP. Chemical and enzymological estimation of polyphosphates and ATP, respectively, indicated the absence of polyphosphates and the presence of ATP in trace amounts in the seed extracts. Immunolocalization using antibodies raised against KBPAP was unsuccessful because of the non-specificity of the antiserum toward glycoproteins. Using histoenzymological methods with ATP as a substrate, we could localize KBPAP exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons. KBPAP activity was not detected in the embryo. In vitro experiments indicated that pectin, a major component of the cell wall, significantly altered the kinetic properties of KBPAP. The substrate profile and localization suggest that KBPAP may have a role in mobilizing organic phosphates in the soil during germination.
已知磷酸酶在植物的磷周转中起着关键作用。然而，由于对其体内底物和亚细胞定位了解不足，酸性磷酸酶在植物中的具体作用一直难以捉摸。我们研究了从红芸豆（菜豆）中分离出的一种紫色酸性磷酸酶（KBPAP）的生化特性，包括其底物和抑制剂谱。测定了五种底物的动力学参数。我们使用³¹P核磁共振来研究KBPAP的体内底物。分别对多聚磷酸盐和ATP进行化学和酶学测定，结果表明种子提取物中不存在多聚磷酸盐，仅存在痕量的ATP。由于抗血清对糖蛋白的非特异性，使用针对KBPAP产生的抗体进行免疫定位未成功。使用以ATP为底物的组织酶学方法，我们能够将KBPAP专门定位在子叶外周两到三排细胞的细胞壁中。在胚中未检测到KBPAP活性。体外实验表明，细胞壁的主要成分果胶显著改变了KBPAP的动力学特性。底物谱和定位表明，KBPAP可能在种子萌发过程中参与动员土壤中的有机磷。
