IDENTIFICATION OF NA-K+-ATPASE BETA-SUBUNIT IN ALVEOLAR EPITHELIAL-CELLS()

The Na+-K+-ATPase is a heterodimeric plasma membrane protein that cons ists of a catalytic alpha-subunit and a smaller glycosylated beta-subu nit that has not been fully characterized in alveolar epithelial cells (AEC) to date. In this study, we identified the Na+-K+-ATPase beta-su bunit protein in rat AEC and lung membranes using immunochemical techn iques. Rat AEC grown in primary culture and rat lung, brain, and kidne y membranes were solubilized in either 2% sodium dodecyl sulfate (SDS) sample buffer for SDS-polyacrylamide gel electrophoresis or in 1% Non idet P-40 lysis buffer for immunoprecipitation studies. Na+-K+-ATPase beta-subunit was not detected in either AEC or lung membranes on Weste rn blots when probed with a panel of antibodies (Ab) against beta-subu nit isoforms, whereas brain and kidney beta-subunit were recognized as broad similar to 50-kDa bands. AEC, lung, and kidney membranes were i mmunoprecipitated with anti-beta Ab IEC 1/48, a monoclonal Ab that rec ognizes beta-subunit protein only in its undenatured state. The beta-s ubunit was detected in the immunoprecipitate (IP) from kidney membrane s by several different anti-beta-subunit Ab. The beta-subunit was fain tly detectable from AEC and lung IP as a broad similar to 50-kDa band when blotted with the polyclonal anti-beta(1)-subunit Ab SpET but coul d not be detected by blotting with other anti-beta Ab. Treatment of th e IP from kidney, lung, and AEC with N-glycosidase F for 2 h at 37 deg rees C resulted in immunodetection of identical similar to 35 kDa band s when probed with all anti-beta(1) Ab on Western blots. From these re sults, we conclude that rat lung and AEC possess immunoreactive beta-s ubunit 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 cond itions of these experiments, our results suggest that lung beta-subuni t may be glycosylated differently from kidney and other tissues. These differences appear to be due to organ- or cell-specific posttranslati onal processing of the beta(1)-subunit and may result in altered regul ation of sodium pumps in lung compared with other epithelia.