SELF-ASSOCIATION OF THE MOLECULAR CHAPERONE HSC70
SELF-ASSOCIATION OF THE MOLECULAR CHAPERONE HSC70
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DOI：
10.1021/bi00046a037
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发表时间：
1995-11-21
期刊：
影响因子：
2.9
通讯作者：
LADJIMI, MM
中科院分区：
文献类型：
--
作者：
BENAROUDJ, N;BATELIER, G;LADJIMI, MM
分子伴侣HSC 70的自缔合特性已经通过广泛的生物化学和生物物理技术进行了分析。非变性凝胶电泳和交联研究表明，从单体到至少三聚体的多个物种的存在。分子排阻色谱法得到两个重叠峰，一个主峰对应于分子量为单体（70 kDa）的物质，一个次宽峰对应于分子量范围为150-300 kDa的物质。蛋白质的逐步稀释导致单体峰的大小增加，而低聚物峰的大小减少，因此表明存在浓度依赖性化学平衡。沉降速度揭示了三种物质的存在下，其比例依赖于浓度，但其沉降系数，S-20，S-W，4.3，6.6和8.5 S不随浓度而变化，表明一个缓慢平衡的系统。沉降平衡研究证实了这些结果，并显示在低浓度下解离成单体，在高浓度下缔合成二聚体和三聚体。在不同的初始负荷浓度以及不同的转子速度下获得的多个沉降平衡数据集，用单体-二聚体-三聚体缔合模型拟合了一组平衡常数，其中单体-二聚体和二聚体-三聚体的缔合常数分别为K-1-2 = 1.1 × 10（5）M（-1）和K-2-3 = 0.9 × 10（5）M（-1）M（-1）。有趣的是，一个等键的，不确定的关联类型几乎同样好地描述了数据与一个单一的常数1.2 × 10（5）M（-1）。总之，这些结果表明，HSC 70缓慢和可逆的自我协会在解决方案中，可能在一个无限的方式，并可能有影响的伴侣蛋白功能的这种蛋白质。
The self association properties of the molecular chaperone HSC70 have been analyzed by a wide range of biochemical and biophysical techniques. Nondenaturing gel electrophoresis and crosslinking studies show the presence of multiple species going from monomer to at least trimer. Size-exclusion chromatography gives two overlapping peaks, a major one corresponding to species having the molecular mass of monomer (70 kDa) and a minor broad one corresponding to species with a molecular mass range of 150-300 kDa. Progressive dilution of the protein leads to an increase in the size of the monomer peak at the expense of that of the oligomeric peak, thus indicating a concentration-dependent chemical equilibrium. Sedimentation velocity reveals the presence of three species, whose proportions were dependent on concentration, but whose sedimentation coefficients, S-20,S-W, of 4.3, 6.6, and 8.5 S did not vary with concentration, indicative of a slowly equilibrating system. Sedimentation equilibrium studies confirmed these results and showed a dissociation into monomers at low concentrations and an association into dimers and trimers at high concentrations. The multiple sedimentation equilibrium datasets, obtained at various initial loading concentrations as well as different rotor speeds, were fitted to a single set of equilibrium constants by a monomer-dimer-trimer association model in which the association constants for the monomer-dimer and dimer-trimer equilibrium were respectively K-1-2 = 1.1 X 10(5) M(-1) and K-2-3 = 0.9 X 10(5) M(-1). Interestingly, an isodesmic, indefinite type of association describes the data almost equally well with a single constant of 1.2 x 10(5) M(-1). Altogether, these results indicate that HSC70 slowly and reversibly self-associates in solution, likely in an unlimited fashion, and might have implications for the chaperone function of-this protein.