Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein

RTI40 is a 40-42 kDa protein that, within the lung, is specific to the apic al plasma membrane of the rat alveolar type I cell. Type I cells cover grea ter than 95% of the internal surface area of the lung. In this report, we d escribe some of the physical properties of RTI40, and its purification to h omogeneity. By liquid phase isoelectric focusing, the pi of the protein is 3.0 +/- 0.5. In two-dimensional immunoblots, there is a 1.0 pH unit charge train, suggesting post-translational modification of the protein. We have p urified the protein to homogeneity by the following method. A membrane prep aration from perfused rat lungs was extracted with detergent and applied to an ion-exchange column. Immunoreactive fractions from the column were pool ed, dialyzed and further fractionated by reverse phase high performance liq uid chromatography (HPLC), Essentially all the antigenicity was recovered i n one protein peak that was homogeneous both by spectral analysis and silve r-stained polyacrylamide gels. Because the purified protein was N terminus blocked, we cleaved the protein with CNBr and fractionated peptide fragment s by reverse phase HPLC. Fractions were pooled and concentrated. Direct ami no acid sequencing of the major peptide fragment yielded a 15 amino acid pe ptide homologous to a mouse osteoblast protein, OTS-8. Analysis of purified RTI40 shows that the protein contains glycan, some of which is sialic acid . Characterization of RTI40 should facilitate future studies of the functio nal properties of RTI40. (C) 1998 Elsevier Science B.V. All rights reserved .