Guranowski A, Jakubowski H, Holler E
J Biol Chem. 1983 Dec 25;258(24):14784-9.
Enzymatic activity which hydrolyzes diadenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) yielding ADP has been identified in extracts of eubacteria, Escherichia coli and Acidaminococcus fermentans, and of a highly thermophilic archaebacterium, Pyrodictum occultum. Specific Ap4A (symmetric) pyrophosphohydrolase from Escherichia coli K12 has been purified almost 400-fold. The preparation was free of phosphatase, ATPase, phosphodiesterase, AMP-nucleosidase, and adenylate kinase. The Ap4A pyrophosphohydrolase molecular weight estimated by gel filtration is 27,000 +/- 1,000. Activity maximum is at pH 8.3. The Km value computed for Ap4A is 25 +/- 3 microM. The sulfhydryl group(s) is essential for enzyme activity. Metal chelators, EDTA, and o-phenanthroline, inhibit Ap4A hydrolysis; I0.5 values are 3 and 50 microM, respectively. Co2+ is a strong stimulator with an almost 100-fold increase in rate of Ap4A hydrolysis and a plateau in the range of 100-500 microM Co2+, when compared with the nonstimulated hydrolysis. Other transition metal ions, Mn2+, Cd2+, and Ni2+, stimulate by factors of 8, 3.5, and 3.5, respectively, with optimal concentrations in the range 200-500, 2-5, and 4-8 microM, respectively. Zn2+, Cu2+, and Fe2+, up to 30 microM, are without effect and they inhibit at higher concentrations. Mg2+ or Ca2+, in the absence of other divalent metal ions, are weak stimulators (1.5-fold stimulation occurs at 1-2 mM concentration), but act synergistically with Co2+ at its suboptimal concentrations. Stimulation in the presence of 10 microM Co2+ and either 1 mM MgCl2 or CaCl2 increases up to 75-fold. The same degree of synergy is found at 10 microM Co2+ and either 2-5 mM spermidine or 0.5-1.5 mM spermine. Besides Ap4A, bacterial Ap4A pyrophosphohydrolase hydrolyzes effectively Ap5A and Gp4G, and, to some extent, p4A, Ap6A, and Ap3A yielding in each case corresponding nucleoside diphosphate as one of the products.
在真细菌（大肠杆菌和发酵氨基酸球菌）以及一种嗜热古细菌（隐蔽火球菌）的提取物中，已鉴定出能水解二腺苷5′,5″-P1,P4-四磷酸（Ap4A）生成ADP的酶活性。来自大肠杆菌K12的特异性Ap4A（对称）焦磷酸水解酶已被纯化了近400倍。该制剂不含磷酸酶、ATP酶、磷酸二酯酶、AMP核苷酶和腺苷酸激酶。通过凝胶过滤估计的Ap4A焦磷酸水解酶分子量为27,000±1,000。活性最大值在pH 8.3。计算得出的Ap4A的Km值为25±3μM。巯基对于酶活性至关重要。金属螯合剂乙二胺四乙酸（EDTA）和邻菲罗啉可抑制Ap4A水解；I0.5值分别为3和50μM。Co2+是一种强刺激剂，与未受刺激的水解相比，Ap4A水解速率增加近100倍，在100 - 500μM Co2+范围内达到平稳状态。其他过渡金属离子Mn2+、Cd2+和Ni2+的刺激倍数分别为8、3.5和3.5，最佳浓度分别在200 - 500μM、2 - 5μM和4 - 8μM范围内。高达30μM的Zn2+、Cu2+和Fe2+无作用，在较高浓度时会产生抑制作用。在没有其他二价金属离子的情况下，Mg2+或Ca2+是弱刺激剂（在1 - 2 mM浓度下刺激1.5倍），但在Co2+亚最佳浓度时与Co2+协同作用。在存在10μM Co2+和1 mM MgCl2或CaCl2的情况下，刺激作用增加至75倍。在10μM Co2+和2 - 5 mM亚精胺或0.5 - 1.5 mM精胺的情况下也发现了相同程度的协同作用。除了Ap4A外，细菌Ap4A焦磷酸水解酶还能有效水解Ap5A和Gp4G，在某种程度上还能水解p4A、Ap6A和Ap3A，每种情况下都会产生相应的核苷二磷酸作为产物之一。
