Lepock J R, Frey H E, Ritchie K P
Guelph-Waterloo Program for Graduate Work in Physics, University of Waterloo, Ontario, Canada.
J Cell Biol. 1993 Sep;122(6):1267-76. doi: 10.1083/jcb.122.6.1267.
There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.
有间接证据表明，在高温热休克期间细胞内会发生蛋白质变性，且变性或受损的蛋白质是热休克反应的主要诱导物。然而，关于热休克期间正常细胞蛋白质的变性程度，尚无直接证据。差示扫描量热法（DSC）是监测蛋白质变性或解折叠的最直接方法。由于所测量的基本参数是热流，DSC可用于检测和定量复杂结构（如分离的细胞器甚至完整细胞）中的吸热转变。从分离的大鼠肝细胞、肝匀浆和中国仓鼠肺V79成纤维细胞中获得了具有共同特征的DSC图谱。观察到五个主要转变（A - E），其中几个可分解为亚组分，转变温度（Tm）为45 - 98℃。起始温度约为40℃，但有些转变可能低至37 - 38℃。除了作为热休克蛋白合成的主要信号外，关键蛋白质的失活可能导致细胞死亡。关键靶点分析表明，V79细胞杀伤的限速步骤是A转变内Tm = 46℃的一种蛋白质的失活。来自大鼠肝脏的分离微粒体膜、线粒体、细胞核和胞质部分具有不同的DSC图谱，这些图谱对完整肝细胞图谱中的不同峰有贡献。因此，完整细胞的DSC图谱似乎是所有亚细胞器和组分图谱的总和。分离细胞器中存在吸热转变有力地证明它们是由蛋白质变性引起的。每个分离的细胞器在接近40℃时有变性起始点，并且含有在关键靶点预测的Tm（46℃）变性的热不稳定蛋白质。在任何温度下的变性程度可以通过分数热焓大致估算。在以1℃/min扫描至45℃并立即冷却（一种相对温和的热休克）后，在肝细胞、V79细胞以及除细胞核外的分离细胞器中发现估计的变性分数为4 - 7%，由于染色质的高热稳定性，细胞核仅发生1%的变性。因此，热不稳定蛋白质似乎存在于所有细胞器和组分中，即使是轻度热休克后，蛋白质变性也是广泛且大量的。
