17] Modified membrane filtration methods for ligand binding on ATP-driven pumps during ATP hydrolysis

Abstract

Publisher Summary This chapter focuses on the modified membrane filtration methods for ligand binding on ATP-driven pumps during ATP hydrolysis. The study of ligand binding on the ATP-driven pumps during ATP hydrolysis is important to clarify the molecular mechanism of active transport of cation. Direct measurement of the ligand binding on the pumps has been done using equilibrium dialysis or ultracentrifugation. These are inappropriate for measurement of the ligand binding during ATP hydrolysis, because they require long periods for measurement. A conventional membrane filtration is a relatively rapid and sensitive method for detecting the ligand binding in the course of enzyme turnover, as long as two conditions are met. (1) Dissociation is sufficiently slow that the bound ligand is not lost when the enzyme is collected on a filter. (2) Affinity of the enzyme for ligand is satisfactorily high for accurate measurement. The amount of monovalent cations bound to the Na + , K + -ATPase during ATP hydrolysis is measured using the double-membrane filtration method. The double-membrane filtration method is applicable for measuring the Ca 2+ binding during ATP hydrolysis, which provides us with useful information about the SR Ca 2+ -ATPase reaction.