Investigation of electrogenic partial reactions in detergent-solubilized Na,K-ATPase

Abstract

A method to investigate electrogenic partial reactions in the pump cycle of membrane-bound P-type ATPases with electrochromic fluorescent dyes has been extended to detergent-solubilized native and purified recombinant Na,K-ATPase. As a first step, it has been shown here that the function and ion-binding properties of the detergent-soluble and membrane-bound rabbit renal Na,K-ATPase are not significantly different. Thus, the new assay overcomes a previous limitation of the styryl dye method, in that that the protein need not be embedded in a membrane at a high density. As an example of an application of this method, transport properties of recombinant Na,K-ATPase purified from yeast cells have been studied. We have investigated and compared Na+ and K+-binding properties of purified detergent-soluble human α1/his-β1 and α2/his-β1 isoforms of the sodium pump. The only significant difference found with respect to ion binding between both isoforms is an almost three-fold lower affinity for K+ binding in the E2P state of the α2/his-β1 isoform. This technique should be readily applicable to various other P-type ATPases, or transport proteins such as carriers or ion channels that can be purified in a detergent-soluble active form.