Cholesterol increases the thermal stability of the Ca 2+/Mg 2+-ATPase of cardiac microsomes

Abstract

The effect of membrane cholesterol on the thermal inactivation of Ca 2+/Mg 2+-ATPase activity of bovine cardiac microsomes 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 Ca 2+/Mg 2+-ATPase activity and thermal denaturation were shifted to higher temperatures in microsomes enriched with cholesterol (37 ± 5 μg cholesterol/mg protein, cholesterol/phospholipid molar ratio 0.31) compared to control microsomes (15 ± 3 μg 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 ( T I, temperature of half activity) were 58.9 ± 0.3°C for control membranes and 59.9 ± 0.1°C for cholesterol-enriched membranes, respectively. Second, measuring ATPase activity as a function of time at constant temperature, where T I values of 57.6 ± 0.5°C and 59.2 ± 0.5°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°C. This T 1 2 , which is a measure of conformational stability, correlates with the T I for Ca 2+/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.