Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium.
Enthalpic and entropic contributions to actin stability: calorimetry, circular dichroism, and fluorescence study and effects of calcium.
复制标题
焓和熵对肌动蛋白稳定性的贡献：量热法、圆二色性、荧光研究和钙的影响。
DOI：
10.1021/bi00453a040
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Tsong,TY
中科院分区：
文献类型：
--
作者：
Bertazzon,A;Tian,GH;Lamblin,A;Tsong,TY
材料和方法肌动蛋白纯化。按照Pardee和Spudich（1982）的描述，从新西兰白色雄性家兔肌肉的丙酮粉末中纯化肌动蛋白。对于涉及热变性的任何测量，通过用HEPES（HEPES，2 mM，pH 8.0; NaATP，0.2 mM; CaCl 2，0.2 mM;和巯基乙醇，0.5 mM）替换Tris-HCl来修改该程序。通过将NaATP浓度提高至1 mM并向先前的缓冲液中添加50 mM KCl和2 mM MgCl 2，G-肌动蛋白聚合为F-肌动蛋白。通过使用电子显微镜监测细丝的形成。如Laemmli（1970）所述，使用Bio-Rad miniprotean II系统，通过SDS-聚丙烯酰胺凝胶电泳（PAGE）检查蛋白质的纯度。通过G-肌动蛋白的UV吸收（λ 290 = 6.3）测定蛋白质浓度。如Lowry等人所述，通过测定氮含量获得F-肌动蛋白的浓度。（1951年）。差示扫描量热法。DSC用通过A/D转换器（DT 2801板）连接到AT & Tpc 6300个人计算机的MC-2扫描量热计（Amherst，MA）进行。该软件由制造商作为数据采集包（EMF软件）提供。该软件包的数据分析基于Freire和Biltonen（1978 a-c）的去卷积程序。在这个过程中，系统的配分函数直接由DSC数据得到，DSC谱可以分解为多个独立的或顺序的两态过程。量热计单元的容量为1.241 mL，并且使用了所有可用的加热速率，在0.16和1.5 K/min之间。将蛋白质相对于参比缓冲液透析过夜，并使用1.2至13 mg/mL的浓度范围，对焓和Tm无明显影响。将基线绘制为从T）到转变的Tf的直线，这两个温度分别为初始温度和最终温度，用于从DSC曲线进行分析（Takahashi等人，1981年）。范特霍夫焓通过配分函数Q（Privalov和Potekhin，1986）计算：
Materials and Methods Actin Purification. Actin was purified from acetone powder of muscle from New Zealand white male rabbits as described by Pardee and Spudich (1982). The procedure was modified by replacing Tris-HCl with HEPES (HEPES, 2 mM, pH 8.0; NaATP, 0.2 mM; CaCl2, 0.2 mM; and mercaptoethanol, 0.5 mM) for any measurement involving heat denaturation. G-Actin was polymerized to F-actin by raising the concen-tration of NaATP to 1 mM and by adding 50 mM KC1 and 2 mM MgCl2 to the previous buffer. Formation of filaments was monitored by using electron microscopy. The purity of the protein was checked by SDS-polyacrylamide gel electrophoresis (PAGE), as described by Laemmli (1970), using a Bio-Rad miniprotean II system. The protein concentration was determined by UV absorption (Ģ2go= 6.3) for G-actin. The concentration of F-actin was obtained by determination of the nitrogen content as described by Lowry et al.(1951). Differential Scanning Microcalorimetry. DSC was per-formed with a MC-2 scanning calorimeter (Amherst, MA) interfaced to an AT & Tpc 6300 personal computer through an A/D converter (DT 2801 board). The software was provided by the manufacturer as a data acquisition package (EMF software). The data analysis of this package is based on the deconvolution procedure of Freire and Biltonen (1978a—c). In this procedure, the partition function of the system is directly obtained from DSC data, and a DSC spectrum can beresolved into either multiple independent or sequential two-state pro-cesses. The capacity of the calorimeter cell is 1.241 mL, and all the available heating rates, between 0.16 and 1.5 K/min, were used. Proteins were dialyzed overnight against the reference buffer, and concentrations ranging from 1.2 to 13 mg/mL were used, with no noticeable effect on the enthalpy and Tm. Baselines were drawn as a straight line from the 7) to the Tf of the transition, thetwo temperatures being the initial and the final temperatures, respectively, for analysis from a DSC curve (Takahashi et al., 1981). The van’t Hoff enthalpy was calculated by means of the partition function Q (Privalov & Potekhin, 1986): AHyli=