The class C acid phosphatase of Helicobacter pylori is a 5′ nucleotidase
The class C acid phosphatase of Helicobacter pylori is a 5′ nucleotidase
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DOI：
10.1016/j.pep.2003.08.020
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发表时间：
2004-01-01
影响因子：
1.6
通讯作者：
Calcutt, MJ
中科院分区：
文献类型：
--
作者：
Reilly, TJ;Calcutt, MJ
幽门螺杆菌的hppA基因产物的纯化和表征研究的结果证实其为C类酸性磷酸酶。hppA基因在H. pylori ATCC菌株49503经PCR扩增和修饰，克隆到pET 21 b中，并在大肠杆菌中过量表达。将重组蛋白从膜中释放出来，并用阳离子交换和Ni螯合层析纯化（16 x）至表观均一性，得到总起始活性的39%回收率。经SDS-PAGE分析，重组酸性磷酸酶的变性分子量为24 kDa。粗品和纯化后的样品中的磷酸酶活性可以复性，并在SDS-PAGE后检测。通过Superdex 75凝胶过滤层析，重组酶的天然分子量约为72 kDa。磷酸盐和酒石酸盐对磷酸酶活性的影响不大，而钒酸盐、EDTA和EDTA对酶活性有显著的抑制作用。磷酸单酯酶水解对硝基苯基磷酸盐（pNPP）以及其他底物的活性在二价阳离子包括Cu 2+、Ni 2+、Co 2+和Mg 2+的存在下增强。重组HppA具有窄的底物特异性，对芳基磷酸酯具有最高的活性，对5'核苷单磷酸酯具有显著的活性。嘌呤和嘧啶5 ′-磷酸的最适pH分别为4.6和5.2。发现5'核苷单磷酸的亲和常数为0.5-1 mM。本研究的结果证实了HppA包含在C类酸性磷酸酶中，并将其鉴定为5'核苷酸酶。(C)2003年爱思唯尔公司All rights reserved.
The results from purification and characterization studies of the hppA gene product of Helicobacter pylori confirm its identification as a class C acid phosphatase. The hppA gene of H. pylori ATCC strain 49503 was amplified and modified by PCR, cloned into pET21b, and overexpressed in Escherichia coli. The recombinant protein was liberated from membranes and purified (16x) to apparent homogeneity with cation exchange and Ni-chelate chromatography resulting in a recovery of 39% of total starting activity. The recombinant acid phosphatase exhibited a denatured molecular mass of 24 kDa by SDS-PAGE. Phosphatase activity in both crude and purified samples could be renatured and detected after SDS-PAGE. The native molecular mass of recombinant enzyme was approximately 72 kDa by gel filtration chromatography on Superdex 75. While phosphate and tartrate had little effect on phosphatase activity, molybdate, vanadate, and EDTA had significant inhibitory effects on enzymatic activity. Phosphomonoesterase activity for hydrolysis of p-nitrophenylphosphate (pNPP) as well as other substrates was enhanced in the presence of divalent cations including Cu2+, Ni2+, Co2+, and Mg2+. Recombinant HppA had narrow substrate specificity with highest activity for arylphosphates and significant activity for 5' nucleoside monophosphates. The pH optimum for enzyme activity was 4.6 and 5.2 for purine and pyrimidine 5' monophosphates, respectively. The affinity constants for the 5' nucleoside monophosphates were found to be 0.5-1 mM. Results from this study confirm HppA inclusion in the class C acid phosphatases and led to its identification as a 5' nucleotidase. (C) 2003 Elsevier Inc. All rights reserved.