Abstract
A method for investigating 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 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 alpha1/his-beta1 and alpha2/his-beta1 isoforms of the sodium pump. The only significant difference found with respect to ion binding between both isoforms is an almost 3-fold lower affinity for K+ binding in the E2P state of the alpha2/his-beta1 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.
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