TY - JOUR
T1 - Molecular dissection of functional domains of the E1E2-ATPase using sodium and calcium pump chimeric molecules
AU - Luckie, D. B.
AU - Lemas, V.
AU - Boyd, K. L.
AU - Fambrough, D. M.
AU - Takeyasu, K.
N1 - Funding Information:
This work was supported by National Institutes of Health grants GM44373 (to K. Takeyasu), HL27867 (to G. Inesi) and NS23241 (to D. M. Fambrough). D. B. Luckie and K. L. Boyd were supported by National Institutes of Health Training Grant T32-HL07284. K. Takeyasu is an Established Investigator of the American Heart Association and a member of the Cancer Center and the Diabetes Center at the University of Virginia.
PY - 1992
Y1 - 1992
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0026725345&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0026725345&partnerID=8YFLogxK
U2 - 10.1016/S0006-3495(92)81807-6
DO - 10.1016/S0006-3495(92)81807-6
M3 - Article
C2 - 1318102
AN - SCOPUS:0026725345
SN - 0006-3495
VL - 62
SP - 220
EP - 227
JO - Biophysical journal
JF - Biophysical journal
IS - 1
ER -