1856 IDENTIFICATION OF THE INTRACELLULAR PRECURSOR TO RAT PULMONARY SURFACTANT APOLIPOPROTEIN(S) A:

The major protein components of rat pulmonary surfactant are acidic proteins, pI =4. 3-4. 8, referred to as Apo (s) A1, Mr=26,000; A2, Mr=32,000; and A3, Mr=38,000. Although it is widely accepted that surfactant is synthesized and secreted into the airway by alveolar Type II epithelial cells, the molecular characteristics of intracellular Apo (s) A precursor (s) have not been established. Toward this end poly (A) mRNA was isolated from adult rat lungs. In vitro translation resulted in mRNA-dependent incorporation of 35S-methionine into TCA-precipitable proteins. Immunoprecipitation of these translation products, with rabbit antisera directed against rat Apo (s) A, identified a protein with Mr=26,000; this protein was not precipitated by non-immune rabbit sera. Specificity of the immunoprecipitated protein was verified by competition experiments: addition of rat lung Apo A completely inhibited immunoprecipitation of Mr=26,000; addition of BSA or DPPC had no effect. Further, Mr=26,000 was not detected in in vitro translated rat liver poly (A)+ mRNA. Two-dimensional SDS-PAGE demonstrated that Mr=26,000, pI=4.3, co-migrated with Apo A1, from rat lung lavage and also with protein immunoprecipitated from 35S-methionine-labelled Type II epithelial cells. Addition of tuni-camycin to Type II cell cultures resulted in appearance of Apo A1, but not Apo (s) A2. and A3. Peptide maps of lung lavage Apo (s) A1, A2 and A3 were identical. Collectively, these observations demonstrate that Mr=26,000 is the intracellular precursor to rat pulmonary surfactant Apo (s) A and that larger molecular weight forms result from extensive N-linked glycosylation of the primary translation product.