Matrix deposition and extracellular processing of newly synthesized collagens in the isolated perfused rat lung.
We have examined the matrix deposition and proteolytic processing of newly synthesized interstitial and basement membrane collagens in the isolated perfused adult rat lung. Isolated, perfused, and ventilated lungs were labeled for up to 4 h with radiolabeled proline. Collagens were partially purified from homogenates by salt fractionation and ion exchange chromatography and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The major collagenous species were identified as types I, III, and IV collagen by peptide mapping and indirect immunoprecipitation assays. Whereas extraction with neutral salts recovered radiolabeled types I and III collagen, extraction of the neutral salt residue with 2 M guanidine-HCl preferentially recovered types III and IV collagen. Reextraction of the guanidine-HCl residue in the presence of dithiothreitol selectively recovered type IV procollagen (PC) and covalently cross-linked aggregates of type IV chains. In pulse-chase experiments we observed extensive conversion of type I PC to collagen during a 4-h chase. Although type III PC was efficiently converted to p-collagen, only small amounts of fully processed chains were identified. Type IV PC did not undergo detectable proteolytic processing. The isolated perfused rat lung should prove useful for further studies of lung collagen metabolism.
Crouch
E C
EC
Department of Pathology, Jewish Hospital, St. Louis, MO 63110.
Moxley
M A
MA
Longmore
W J
WJ
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005109:Extracellular Matrix / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008168:Lung / Q000378:metabolism*; D008297:Male; D010449:Peptide Mapping; D010477:Perfusion; D011392:Proline / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051381:Rats; D011919:Rats, Inbred Strains
D003094:Collagen; D011392:Proline