Investigation of electrogenic partial reactions in detergent-solubilized Na,K-ATPase.
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.
Habeck
Michael
M
Department of Biology, University of Konstanz, 78464 Konstanz, Germany.
Cirri
Erica
E
Katz
Adriana
A
Karlish
Steven J
SJ
Apell
Hans-Jürgen
HJ
D000818:Animals; D020134:Catalytic Domain; D003902:Detergents; D006801:Humans; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D010766:Phosphorylation; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012995:Solubility; D013050:Spectrometry, Fluorescence
D003902:Detergents; D007527:Isoenzymes; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase