Protection of the membrane calcium adenosine triphosphatase by cholesterol from thermal inactivation

There is correlative evidence that one mechanism of cellular thermoresistance is an increased level of membrane cholesterol. The hypothesis that cholesterol protects membrane proteins from thermal inactivation was tested using Ca-ATPase as a model. The intracellular Ca2+- and Mg2+-dependent ATPase from muscle sarcoplasmic reticulum was reconstituted into lipid mixtures containing different amounts of cholesterol [cholesterol/phospholipid molar ratio (C/PL) = 0.1 or 0.3]. The rate of thermal inactivation of calcium uptake activity of the reconstituted vesicles with C/PL = 0.3 was found to be significantly lower than those with C/PL = 0.1 in the temperature range 43-47 degrees C where hyperthermic cell killing occurs. At 43 degrees C, this is equivalent to a 3 degrees C temperature shift. ATP hydrolysis of Ca-ATPase was found to be substantially heat resistant in reconstituted vesicles with C/PL = 0.1 or 0.3. Glycerol (10%) protects while ethanol (2.5%) and the local anesthetics dibucaine, tetracaine, and procaine sensitize the thermal inactivation of calcium uptake. To investigate the molecular mechanisms of thermal inactivation and cholesterol protection, the responses of the physical state of the lipid and protein conformation to hyperthermic sensitizers and protector were monitored using fluorescent and spin label probes and circular dichroism, respectively. The calcium uptake inactivation appears to be due to a direct thermotropic conformational change (denaturation) of the protein. Cholesterol raises the temperature of inactivation, as does glycerol, while ethanol and the local anesthetics lower it.