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首页> 外文期刊>The FEBS journal >ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli
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ADPase activity of recombinantly expressed thermotolerant ATPases may be caused by copurification of adenylate kinase of Escherichia coli

机译：重组表达的耐热ATPase的ADPase活性可能是由大肠杆菌腺苷酸激酶的共纯化引起的

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摘要

Except for apyrases, ATPases generally target only the gamma-phosphate of a nucleotide. Some non-apyrase ATPases from thermophilic microorganisms are reported to hydrolyze ADP as well as ATP, which has been described as a novel property of the ATPases from extreme thermophiles. Here, we describe an apparent ADP hydrolysis by highly purified preparations of the AAA+ ATPase NtrC1 from an extremely thermophilic bacterium, Aquifex aeolicus. This activity is actually a combination of the activities of the ATPase and contaminating adenylate kinase (AK) from Escherichia coli, which is present at 1/10,000 of the level of the ATPase. AK catalyzes conversion of two molecules of ADP into AMP and ATP, the latter being a substrate for the ATPase. We raise concern that the observed thermotolerance of E. coli AK and its copurification with thermostable proteins by commonly used methods may confound studies of enzymes that specifically catalyze hydrolysis of nucleoside diphosphates or triphosphates. For example, contamination with E. coli AK may be responsible for reported ADPase activities of the ATPase chaperonins from Pyrococcus furiosus, Pyrococcus horikoshii, Methanococcus jannaschii and Thermoplasma acidophilum; the ATP/ADP-dependent DNA ligases from Aeropyrum pernix K1 and Staphylothermus marinus; or the reported ATP-dependent activities of ADP-dependent phosphofructokinase of P. furiosus. Purification methods developed to separate NtrC1 ATPase from AK also revealed two distinct forms of the ATPase. One is tightly bound to ADP or GDP and able to bind to Q but not S ion exchange matrixes. The other is nucleotide-free and binds to both Q and S ion exchange matrixes.
机译：除无主酶外，ATPase通常仅靶向核苷酸的γ-磷酸。据报道，来自嗜热微生物的一些非磷酸酶ATPase水解ADP和ATP，这被描述为来自极端嗜热菌的ATPase的新特性。在这里，我们描述了由高度嗜热的细菌Aquifex aeolicus的AAA + ATPase NtrC1的高度纯化制备而成的明显ADP水解。该活性实际上是ATPase和污染大肠杆菌的腺苷酸激酶（AK）活性的组合，其含量为ATPase的1 / 10,000。 AK催化ADP的两个分子向AMP和ATP的转化，后者是ATPase的底物。我们引起关注的是，观察到的大肠杆菌AK的耐热性及其通过常用方法与热稳定蛋白的共纯化可能会混淆对特异性催化核苷二磷酸或三磷酸水解的酶的研究。例如，大肠杆菌AK污染可能是造成激烈热球菌，霍里热球菌，詹氏甲烷球菌和嗜酸嗜热球菌ATPase伴侣蛋白的ADPase活性的原因。 Aeropyrum pernix K1和Staphylothermus marinus的ATP / ADP依赖性DNA连接酶;或据报道，ADP依赖性磷酸果糖磷酸果糖激酶的ATP依赖性活性。分离NtrC1 ATPase和AK的纯化方法也揭示了ATPase的两种不同形式。一个与ADP或GDP紧密结合，并且能够与Q结合，但不能与S离子交换基质结合。另一个不含核苷酸，并与Q和S离子交换基质结合。

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