MEMBRANE-BOUND NA-ATPASE AS THE CARDIAC GLYCOSIDE RECEPTOR - A THERMOCHEMICAL CHARACTERIZATION(,K+)

DSC studies are carried out to characterize Na+,K+-ATPase isolated fro m pig kidney in its natural membrane environment as well as in its pur ified state upon detergent treatment. The transition temperatures of t he investigated thermal protein unfolding process, observed between 43 and 64.5 degrees C, depend on the local membrane environment as well as on pH. In addition, the transition temperature is significantly inc reased upon binding of different cations and ligands which are known t o interact with the enzyme. Evidence for a lipid phase transition arou nd 23 degrees C in the original biological membrane is reported.The ap plication of a calorimeter equipped with removable cells appears to be more suitable for the investigation of this type of membrane sample t han an instrument with fixed capillary cells. Filling the sample capil lary cell with an usual syringe, consisting of a long and thin needle, can influence the experimental results. Na+,K+-ATPase acts as the rec eptor for cardiac glycoside binding. The thermodynamic parameters of t his binding process are determined by titration calorimetry. The bindi ng of ouabain, as a typical example, is unusually slow and is enthalpy driven. The enthalpy change upon binding enthalpy is -75 kJ mol(-1) a t 25 degrees C. The surprisingly low stoichiometric coefficient result ing from an evaluation based on a simple one step binding model, is in terpreted in terms of a dimeric receptor unit.