title: The anion exchanger and Na+K(+)-ATPase interact with distinct sites on ankyrin in in vitro assays.
abstract: This report demonstrates that the high affinity binding of ankyrin to two well characterized ankyrin-binding proteins, the erythrocyte anion exchanger and kidney Na+K(+)-ATPase, requires interaction of these proteins with unique sites on the ankyrin molecule. Binding of 125I-labeled erythrocyte ankyrin and ankyrin proteolytic domains was measured to the anion exchanger and Na+K(+)-ATPase incorporated into phosphatidylcholine liposomes. 125I-Labeled ankyrin associated with both anion exchanger and Na+K(+)-ATPase liposomes with a high affinity (KD ranging from 10 to 25 nM), and a capacity approaching 1 mol of ankyrin/2 mol of ATPase and 1 mol of ankyrin/8 mol of anion exchanger. The 43 kDa cytoplasmic domain of the erythrocyte anion exchanger inhibited binding of ankyrin to both the anion exchanger and Na+K(+)-ATPase liposomes with a 50% reduction at approximately 90 nM for both proteins. Further binding experiments using proteolytic domains derived from ankyrin demonstrated the following differences between the anion exchanger and Na+K(+)-ATPase in interactions with ankyrin: 1) 125I-Labeled Na+K(+)-ATPase associated with both the 89-kDa domain as well as the spectrin binding domain of ankyrin, while the anion exchanger only associated with the 89-kDa domain. 2) The 125I-labeled 89-kDa domain of ankyrin associated with Na+K(+)-ATPase liposomes with at least a 20-fold lower affinity compared with intact ankyrin while this domain associated with the anion exchanger with a 2-3-fold increase in affinity compared with intact ankyrin. 3) The 125I-labeled spectrin-binding domain of ankyrin associated with the Na+K(+)-ATPase liposomes to at least an 8-fold greater extent than to anion exchanger liposomes. The data are consistent with an independent acquisition of high affinity ankyrin binding activity for the anion exchanger and Na+K(+)-ATPase proteins through a convergent evolutionary process.
authors.affiliation: Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: J Q
authors.initials: JQ
authors.lastname: Davis
authors.forename: V
authors.initials: V
authors.lastname: Bennett
mesh_terms: D000818:Animals; D017487:Ankyrins; D001665:Binding Sites; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000378:metabolism; D006801:Humans; D007679:Kidney Medulla / Q000201:enzymology; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification / Q000378:metabolism*; D013049:Spectrin / Q000378:metabolism
chemical_list: D017487:Ankyrins; D001798:Blood Proteins; D008081:Liposomes; D008565:Membrane Proteins; D013049:Spectrin; D000254:Sodium-Potassium-Exchanging ATPase