Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro
Zn2+ promotes the self-association of human immunodeficiency virus type-1 integrase in vitro
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DOI：
10.1021/bi961849o
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发表时间：
1997-01-07
期刊：
影响因子：
2.9
通讯作者：
Han, MK
中科院分区：
文献类型：
--
作者：
Lee, SP;Xiao, JM;Han, MK
最近已经证明，纯化的人免疫缺陷病毒1型（HIV-I）整合酶的Mg 2+依赖性3 ′-加工活性被外源Zn 2+的加入所刺激[Lee，S. P.，& Han，M. K.（1996）Biochemistry 35，3837-3844]。假设这种激活是由整合酶自结合引起的。在这份报告中，我们检查Zn 2+含量纯化的HIV-1整合酶的原子吸收光谱法和应用的巯基修饰试剂，对-（羟基汞）苯磺酸盐，与金属显色指示剂，4-（2-吡啶偶氮）间苯二酚。我们发现，HIV-1整合酶的Zn 2+含量变化从0.1至0.92当量的Zn 2+每个单体取决于蛋白质纯化的条件。在体外活性测定，时间分辨荧光发射各向异性，和凝胶过滤色谱分析都表明，EDTA产生的apoprotein主要是单体和活性较低的Mg 2+。此外，沉降平衡的研究表明，重组的脱辅基蛋白与Zn 2+的结果在一个单体-四聚体-八聚体过渡。这些结果表明，Zn 2+促进构象与增强的寡聚化，从而刺激。这也可能意味着，在Zn 2+和Mg 2+的存在下，整合酶的体外活性需要大于二聚体（四聚体和可能的八聚体）的多聚体。然而，值得注意的是，Zn 2+的含量并没有显着影响体外与Mn 2+的3 '-加工和链转移反应。
It has been recently demonstrated that the Mg2+-dependent 3'-processing activity of purified human immunodeficiency virus type-1 (HIV-I) integrase is stimulated by thr addition oi exogenous Zn2+ [Lee, S. P., & Han, M. K. (1996) Biochemistry 35, 3837-3844]. This activation was hypothesized to result from integrase self-association. In this report, we examine the Zn2+ content of purified HIV-1 integrase by atomic absorption spectroscopy and by application of a thiol modification reagent, p-(hydroxymercuri)benzenesulfonate, with a metallochromic indicator, 4-(2-pyridylazo)resorcinol. We find that the Zn2+ content of HIV-1 integrase varies from 0.1 to 0.92 equiv of Zn2+ per monomer depending on the conditions of protein purification. In vitro activity assays, time-resolved fluorescence emission anisotropy, and gel filtration chromatographic analyses all indicate that EDTA yields an apoprotein which is predominantly monomeric and less active with Mg2+. Further, sedimentation equilibrium studies reveal that reconstitution of the apoprotein with Zn2+ results in a monomer-tetramer-octamer transition. These results suggest that Zn2+ promotes a conformation with enhanced oligomerization and thereby stimulates This may also imply that multimers larger than dimers (tetramers and possibly octamers) are required for in vitro activity of integrase in the presence of Zn2+ and Mg2+. It should be noted, however, that the content of Zn2+ did not significantly affect the 3'-processing and strand transfer reactions with Mn2+ in vitro.