Biochemical and Molecular Characterization of AtPAP26, a Vacuolar Purple Acid Phosphatase Up-Regulated in Phosphate-Deprived Arabidopsis Suspension Cells and Seedlings1
Biochemical and Molecular Characterization of AtPAP26, a Vacuolar Purple Acid Phosphatase Up-Regulated in Phosphate-Deprived Arabidopsis Suspension Cells and Seedlings1
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DOI：
10.1104/pp.106.087171
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发表时间：
2006-09
期刊：
影响因子：
7.4
通讯作者：
Vasko Veljanovski;B. Vanderbeld;V. Knowles;W. Snedden;W. Plaxton
中科院分区：
文献类型：
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作者：
Vasko Veljanovski;B. Vanderbeld;V. Knowles;W. Snedden;W. Plaxton
将拟南芥悬浮细胞在磷饥饿过程中积累的液泡酸性磷酸酶（APase）纯化至均一。最终制备物是紫色APase（PAP），因为其在溶液中显示粉红色（Amax = 520 nm）。它以100 kD的同源二聚体形式存在，由55 kD的糖基化亚基组成，与抗（番茄胞内PAP）-IgG交叉反应。其23个氨基酸的N-末端序列的BLAST分析显示，该PAP是由At 5g 34850（AtPAP 26;拟南芥中的29个PAP基因之一）编码的，并且在AtPAP 26前蛋白易位到液泡期间，从AtPAP 26前蛋白切割出30个氨基酸的信号肽。AtPAP 26与其他植物的直系同源物的序列相似性比与其他拟南芥PAP的序列相似性高得多。AtPAP 26在pH 5.6时表现出最佳活性和广泛的底物选择性。在PI剥夺的细胞中发生的AP酶活性的5倍增加被55-kD抗（番茄PAP或AtPAP 26）-IgG免疫反应性多肽的量的类似增加和细胞内游离Pi浓度的>30倍减少所抵消。半定量逆转录-PCR表明，PI充足，PI饥饿，和PI再供应细胞含有类似量的AtPAP 26转录本。因此，相对于翻译和/或蛋白水解控制，转录控制似乎对AtPAP 26水平几乎没有影响。AP酶活性和AtPAP 26蛋白水平也上调，在芽和根的Pi剥夺拟南芥幼苗。我们假设AtPAP 26从Pi饥饿的拟南芥细胞内P代谢物中除去Pi。由于AtPAP 26也表现出碱性过氧化物酶活性，在活性氧代谢的潜在的额外作用进行了讨论。
A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (Amax = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.