Ortega A, Santiago-García J, Mas-Oliva J, Lepock J R
Department of Biology, University of Waterloo, Ontario, Canada.
Biochim Biophys Acta. 1996 Aug 14;1283(1):45-50. doi: 10.1016/0005-2736(96)00072-7.
The effect of membrane cholesterol on the thermal inactivation of Ca2+/Mg(2+)-ATPase activity of bovine cardiac microsome was measured and compared to the thermal denaturation profiles of the microsomes as measured by differential scanning calorimetry (DSC). Inactivation, defined as loss of activity, and denaturation, defined as conformational unfolding, were irreversible under the conditions used. Both thermal inactivation of Ca2+/Mg(2+)-ATPase activity and thermal denaturation were shifted to higher temperatures in microsomes enriched with cholesterol (37 +/- 5 micrograms cholesterol/mg protein, cholesterol/phospholipid molar ratio 0.31) compared to control microsomes (15 +/- 3 micrograms cholesterol/mg protein, molar ratio 0.12). Thermal inactivation was measured by two methods: first, measuring activity at room temperature as a function of heating to elevated temperatures at 1 K/min, where inactivation temperatures (T1, temperature of half activity) were 58.9 +/- 0.3 degrees C for control membranes and 59.9 +/- 0.1 degrees C for cholesterol-enriched membranes, respectively. Second, measuring ATPase activity as a function of time at constant temperature, where T1 values of 57.6 +/- 0.5 degrees C and 59.2 +/- 0.5 degrees C were determined for control and cholesterol-enriched membranes, respectively. DSC profiles of microsomal membranes consisting of a number of overlapping peaks were obtained. A well resolved component (transition C) was observed with a transition temperature (T 1/2) of 58.2 degrees C. This T 1/2, which is a measure of conformational stability, correlates with the T1 for Ca2+/Mg(2+)-ATPase activity and is 1.9 +/- 0.6 K higher in cholesterol-enriched membranes. Thus, the increased resistance to inactivation appears to be due to increased conformational stability of the protein induced by cholesterol, demonstrating that a change in lipid composition can influence the stability of an integral membrane protein in a natural membrane. The increased stability is of sufficient magnitude to account for the previously observed correlation between cholesterol content and resistance to heat shock in several cell lines.
测定了膜胆固醇对牛心脏微粒体Ca2+/Mg(2+)-ATP酶活性热失活的影响，并将其与通过差示扫描量热法（DSC）测定的微粒体热变性曲线进行了比较。在所使用的条件下，失活（定义为活性丧失）和变性（定义为构象展开）都是不可逆的。与对照微粒体（15±3微克胆固醇/毫克蛋白质，摩尔比0.12）相比，富含胆固醇的微粒体（37±5微克胆固醇/毫克蛋白质，胆固醇/磷脂摩尔比0.31）中Ca2+/Mg(2+)-ATP酶活性的热失活和热变性都向更高温度偏移。通过两种方法测量热失活：第一，在室温下测量活性作为以1 K/分钟加热至升高温度的函数，其中对照膜的失活温度（T1，半活性温度）为58.9±0.3℃，富含胆固醇的膜为59.9±0.1℃。第二，在恒定温度下测量ATP酶活性作为时间的函数，其中对照膜和富含胆固醇的膜的T1值分别为57.6±0.5℃和59.2±0.5℃。获得了由多个重叠峰组成的微粒体膜的DSC曲线。观察到一个分辨率良好的组分（转变C），其转变温度（T 1/2）为58.2℃。这个T 1/2是构象稳定性的一个度量，与Ca2+/Mg(2+)-ATP酶活性的T1相关，并且在富含胆固醇的膜中高1.9±0.6 K。因此，对失活的抗性增加似乎是由于胆固醇诱导的蛋白质构象稳定性增加，表明脂质组成的变化可以影响天然膜中整合膜蛋白的稳定性。增加的稳定性足以解释先前在几种细胞系中观察到的胆固醇含量与耐热休克之间的相关性。
