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This chapter focuses on the occluded Ca2+. Ion-transport enzymes undergo a sequence of reaction steps in which the respective ions and phosphates interact with the enzyme mutually. One enzyme state in the main reaction cycle has been defined as occluded with respect to the ion to be translocated, i.e., the ions bound to the enzyme in this state are slowly exchanged with free ions in the medium. In order to measure the occluded ions bound to the enzyme, free ions are removed from assays as rapidly as possible. Several procedures have been applied to studies of the sodium-potassium and the calcium pump. The centrifuge column procedure is the most suitable method to identify occluded Ca2+-binding site and the corresponding phosphoprotein intermediates. The procedure is rather simple and the sensitivity is high. For extended use of this method, the following precautions should be observed. First, when membranous vesicular preparations are used, the membranes should be completely permeable to the transported solute so that only the bound and not the trapped solute are measured. Second, the rate at which the intermediate decomposes should be relatively slow so that no significant decomposition takes place during the column centrifugation.
