Identification of Na(+)-K(+)-ATPase beta-subunit in alveolar epithelial cells.
The Na(+)-K(+)-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic alpha-subunit and a smaller glycosylated beta-subunit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na(+)-K(+)-ATPase beta-subunit protein in rat AEC and lung membranes using immunochemical techniques. Rat AEC grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Nonidet P-40 lysis buffer for immunoprecipitation studies. Na(+)-K(+)-ATPase beta-subunit was not detected in either AEC or lung membranes on Western blots when probed with a panel of antibodies (Ab) against beta-subunit isoforms, whereas brain and kidney beta-subunit were recognized as broad approximately 50-kDa bands. AEC, lung, and kidney membranes were immunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that recognizes beta-subunit protein only in its undenatured state. The beta-subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-beta-subunit Ab. The beta-subunit was faintly detectable from AEC and lung IP as a broad approximately 50-kDa band when blotted with the polyclonal anti-beta 1-subunit Ab SpET but could not be detected by blotting with other anti-beta Ab. Treatment of the IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 degrees C resulted in immunodetection of identical approximately 35 kDa bands when probed with all anti-beta 1 Ab on Western blots. From these results, we conclude that rat lung and AEC possess immunoreactive beta-subunit protein that is only readily detectable after deglycosylation. Because anti-beta Ab fail to detect the Na(+)-K(+)-ATPase beta-subunit in rat lung or AEC by standard Western blotting techniques under the conditions of these experiments, our results suggest that lung beta-subunit may be glycosylated differently from kidney and other tissues. These differences appear to be due to organ- or cell-specific posttranslational processing of the beta 1-subunit and may result in altered regulation of sodium pumps in lung compared with other epithelia.
Zhang
X L
XL
Will Rogers Institute Pulmonary Research Center, University of Southern California, Los Angeles 90033, USA.
Danto
S I
SI
Borok
Z
Z
Eber
J T
JT
Martín-Vasallo
P
P
Lubman
R L
RL
D000818:Animals; D015153:Blotting, Western; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D005643:Fucose; D006031:Glycosylation; D015151:Immunoblotting; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008358:Mannose; D011233:Precipitin Tests; D011650:Pulmonary Alveoli / Q000166:cytology / Q000201:enzymology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D007527:Isoenzymes; D005643:Fucose; D000254:Sodium-Potassium-Exchanging ATPase; D008358:Mannose