We have studied the thermal inactivation at 37 degrees C of wild type and mutant ChE2 (C310A, F312I, C466A, C310A/F312I, and C310A/C466A) from amphioxus (Branchiostoma floridae) expressed in vitro in COS-7 monkey cells under three sets of conditions: 30 degrees C for 48 h, 30 degrees C for 24 h and 37 degrees C for 24 h, and 37 degrees C for 48 h. We found biphasic denaturation curves for all enzymes and conditions, except wild type and C310A ChE2 expressed at 30 degrees C for 48 h. Generally, single mutants are more unstable than wild type, and the double mutants are even more unstable. We propose a model involving stable and unstable conformations of the enzymes to explain these results, and we discuss the implications of the model. We also found a correlation between the melting temperature of the ChEs and the rates at which they denature at 37 degrees C, with the denaturation of the unstable conformation dominating the relationship. Reversible cholinergic inhibitors protect the ChEs from thermal denaturation, and in some cases produce monophasic denaturation curves; we also propose a model to explain this stabilization.

中文翻译：

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来自河豚的野生型和突变重组乙酰胆碱酯酶的热变性：体外表达温度和可逆抑制剂的影响。

我们研究了在三组 COS-7 猴细胞中体外表达的来自 amphioxus (Branchiostoma floridae) 的野生型和突变型 ChE2（C310A、F312I、C466A、C310A/F312I 和 C310A/C466A）在 37 摄氏度下的热灭活条件：30℃48小时，30℃24小时，37℃24小时，37℃48小时。我们发现所有酶和条件的双相变性曲线，除了野生型和 C310A ChE2 在 30 摄氏度表达 48 小时。一般来说，单突变体比野生型更不稳定，双突变体更不稳定。我们提出了一个涉及酶的稳定和不稳定构象的模型来解释这些结果，并讨论了该模型的含义。我们还发现了 ChE 的熔化温度与它们在 37 摄氏度时变性的速率之间存在相关性，不稳定构象的变性主导了这种关系。可逆胆碱能抑制剂保护 ChE 免受热变性，在某些情况下会产生单相变性曲线；我们还提出了一个模型来解释这种稳定性。