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首页> 外文期刊>Journal of Virology >Protein Kinase Activity from Vaccinia Virions: Solubilization and Separation into Heat-Labile and Heat-Stable Components
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Protein Kinase Activity from Vaccinia Virions: Solubilization and Separation into Heat-Labile and Heat-Stable Components

机译：来自疫苗病毒藻的蛋白激酶活性：溶解和分离成热稳定和热稳定的组分

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摘要

A protein kinase was solubilized from whole vaccinia virions by using a solution containing deoxycholate, dithiothreitol, and sodium or potassium chloride. The released enzyme was completely dependent on Mg2+ and was greatly stimulated by added basic proteins such as protamine or histones. Dithiothreitol was also stimulatory, whereas GTP, CTP, UTP, and Pi at concentrations equimolar with ATP had little or no effect. Attempts to purify the protein kinase were initially unsuccessful, leading us to consider that either the enzyme was extremely labile or that two readily separable components were required for activity. The observation that the material extracted with NP-40 detergent during the preparation of viral cores stimulated the protein kinase activity of the intact cores supported the second possibility. As the protein kinase, now solubilized from viral cores, was passed through successive DEAE-cellulose columns, it became increasingly dependent for activity on addition of the NP-40 extract. A 30- to 40-fold stimulation of protein kinase activity, which afforded recovery of essentially all starting activity, could be effected by addition of the NP-40 extract to the partially purified enzyme. The NP-40 extract was shown to contain a heat stable, trypsin-sensitive protein, whose action could not be duplicated by cyclic nucleotides.
机译：通过使用含有脱氧胆酸盐，二硫醇和氯化钠或氯化钾的溶液，从全部疫苗病毒中溶解一种蛋白激酶。释放的酶完全依赖于Mg 2 + ，并通过添加碱性蛋白质如protamine或组蛋白，大大刺激。 Dithiothreitol也是刺激性的，而GTP，CTP，UTP和P I 以ATP的浓度等摩尔几乎没有效果。纯化蛋白激酶的尝试最初是不成功的，导致我们认为酶非常不稳定，或者需要两种易于分离的组分。观察到在制备病毒芯期间用NP-40洗涤剂提取的材料刺激了完整核心的蛋白激酶活性支持第二种可能性。作为蛋白激酶，现在通过患有病毒芯溶解的蛋白质激酶通过连续的脱节纤维素柱，其越来越依赖于添加NP-40提取物的活性。通过向部分纯化的酶加入NP-40提取物来实现30至40倍的蛋白激酶活性的蛋白激酶活性的刺激。显示NP-40提取物被显示为含有热稳定的胰蛋白酶敏感蛋白，其作用不能通过循环核苷酸复制。

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