Razem Fawzi A, Luo Ma, Liu Jin-Hao, Abrams Suzanne R, Hill Robert D
Department of Plant Science, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
J Biol Chem. 2004 Mar 12;279(11):9922-9. doi: 10.1074/jbc.M311064200. Epub 2003 Dec 29.
A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.
一种名为ABAP1的蛋白质由一个新基因（GenBank登录号AF127388）编码，经纯化后显示能特异性结合脱落酸（ABA）。ABAP1蛋白是一种含有WW蛋白相互作用结构域的472个氨基酸的多肽，在大麦糊粉层中受ABA诱导。多克隆抗独特型抗体（AB2）与纯化的ABAP1以及与ABA处理的大麦糊粉层膜组分相关的相应52 kDa蛋白发生交叉反应。在多种单子叶和双子叶植物物种中检测到了ABAP1基因，包括小麦、烟草、苜蓿、豌豆和油菜。重组ABAP1蛋白在中性pH条件下能最佳结合（3）H-（+）-ABA。变性的ABAP1蛋白不结合（3）H-（+）-ABA，牛血清白蛋白也不结合。Scatchard图分析显示最大特异性结合为0.8摩尔ABA/摩尔（-1）蛋白，线性函数r（2） = 0.94，表明存在一个解离常数（K（d））为28×10（-9）米的ABA结合位点。糊粉层质膜中的ABA结合显示最大结合容量为330纳摩尔ABA/克（-1）蛋白，K（d）为26.5×10（-9）米。重组蛋白与质膜的ABA结合解离常数相似，表明质膜组分中的蛋白是ABAP1的天然形式。ABAP1的立体特异性通过以下事实得以确立：ABA类似物和代谢物，包括（-）-ABA、反式ABA、脱落酸、二氢脱落酸和（+）-脱落酸葡萄糖酯，无法取代与ABAP1结合的（3）H-（+）-ABA。然而，两种ABA前体，（+）-ABA醛和（+）-ABA醇，能够取代（3）H-（+）-ABA，这表明ABAP1在C-1位的结构要求并不严格。我们的数据表明ABAP1对ABA具有高结合亲和力。这种相互作用是可逆的，遵循饱和动力学，并且具有立体特异性，因此符合ABA结合蛋白的标准。
