Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules.
Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.
Luckie
D B
DB
Department of Physiology, University of Virginia, Charlottesville 22908.
Lemas
V
V
Boyd
K L
KL
Fambrough
D M
DM
Takeyasu
K
K
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D002645:Chickens; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010042:Ouabain / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism / Q000502:physiology
D011994:Recombinant Proteins; D010042:Ouabain; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium