Identification of na+, k+-atpase b-subunit in alveolar epithelial cells. Zhang, Xiao-Ling, Spencer I. Danto, Zea Borok, Jennifer T. Eber, Pablo Martaen-Vasallo, and Richard L. Lubman. Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, CA 90033 and Laboratorio de Biologaea del Desarrollo, Departamento de Bioquaemica y Biologaea Molecular, Universidad de La Laguna, Avda Astrofaesico S[cedilla]cnchez s/n, 38206 La Laguna, Tenerife, Spain
APStracts 3:0168L, 1996.
The Na+, K+-ATPase is a heterodimeric plasma membrane protein that consists of a catalytic [alpha]-subunit and smaller, glycosylated [beta] subunit that has not been fully characterized in alveolar epithelial cells (AECs) to date. In this study, we identified the Na+, K+-ATPase [beta] subunit protein in rat AECs and lung membranes using immunochemical techniques. Rat AECs grown in primary culture and rat lung, brain, and kidney membranes were solubilized in either 2% SDS sample buffer for SDS-PAGE or 1% NP-40 lysis buffer for immunoprecipitation studies. Na+, K+-ATPase [beta] subunit was not detected in either AECs or lung membranes on western blots when probed with a panel of Abs against [beta] subunit isoforms, while brain and kidney [beta] subunit were recognized as broad 50 kD bands. AECs, 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. [beta] subunit was detected in the immunoprecipitate (IP) from kidney membranes by several different anti-[beta] subunit Abs. [beta] subunit was faintly detectable from AEC and lung IPs as a broad 50 kD 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 IPs from kidney, lung, and AECs with N-glycosidase F for 2 h at 37 C resulted in immunodetection of identical 35 kD bands when probed with all anti -[beta]1 Abs on western blots. From these results, we conclude that rat lung and AECs possess immunoreactive [beta] subunit protein that is only readily detectable after deglycosylation. Since anti-[beta] Abs fail to detect the Na+, K+-ATPase [beta] subunit in rat lung or AECs 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 post -translational processing of the [beta]1-subunit, and may result in altered regulation of Na pumps in lung compared to other epithelia. 

Received 30 May 1996; accepted in final form 3 September 1996.
APS Manuscript Number L158-6.
Article publication pending Am. J. Physiol. (Lung Cell. Mol.
Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 5 November 1996