CHOLESTEROL INCREASES THE THERMAL-STABILITY OF THE CA2+ MG2+-ATPASE OF CARDIAC-MICROSOMES/

The effect of membrane cholesterol on the thermal inactivation of Ca2/Mg2+-ATPase activity of bovine cardiac microsomes was measured and co mpared to the thermal denaturation profiles of the microsomes as measu red by differential scanning calorimetry (DSC), Inactivation, defined as loss of activity, and denaturation, defined as conformational unfol ding, were irreversible under the conditions used, Both thermal inacti vation of Ca2+/Mg2+ ATPase activity and thermal denaturation were shif ted to higher temperatures in microsomes enriched with cholesterol (37 +/- 5 mu g cholesterol/mg protein, cholesterol/phospholipid molar rat io 0.31) compared to control microsomes (15 +/- 3 mu g cholesterol/mg protein, molar ratio 0.12), Thermal inactivation was measured by two m ethods: first, measuring activity at room temperature as a function of heating to elevated temperatures at 1 K/min, where inactivation tempe ratures (T-1, temperature of half activity) were 58.9 +/- 0.3 degrees C for control membranes and 59.9 +/- 0.1 degrees C for cholesterol-enr iched membranes, respectively, Second, measuring ATPase activity as a function of time at constant temperature, where T-1 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 micr osomal membranes consisting of a number of overlapping peaks were obta ined, A well resolved component (transition C) was observed with a tra nsition temperature (T-1/2) of 58.2 degrees C. This T-1/2, which is a measure of conformational stability, correlates with the T-1 for Ca2+/ Mg2+-ATPase activity and is 1.9 +/- 0.6 K higher in cholesterol-enrich ed membranes. Thus, the increased resistance to inactivation appears t o be due to increased conformational stability of the protein induced by cholesterol, demonstrating that a change in lipid composition can i nfluence the stability of an integral membrane protein in a natural me mbrane, The increased stability is of sufficient magnitude to account for the previously observed correlation between cholesterol content an d resistance to heat shock in several cell lines.