Cross-linking in type IV collagen.
Type IV collagen could not be extracted from human placenta using 6M-urea containing 10mM-dithiothreitol, indicating that the type IV molecule is stabilized within the basement membrane by covalent cross-links. However, various forms of type IV collagen molecule were extractable by means of increasingly severe proteolytic conditions. Type IV collagen tetramers ('spiders') lacking only the C-terminal globular region (NC1) were further digested to the 'long-form' 7S fragment and an assortment of helical rod-like molecules derived from the 'leg' region. These molecules were separated by salt fractionation and examined by rotary-shadowing electron microscopy. Isolation of these fractions from placenta which had been reduced with NaB3H4 revealed that both the 7S (N-terminal) and C-terminal regions contained significant proportions of reducible lysine-derived cross-links. These cross-links were shown to be exclusively the stable oxo-imine hydroxylysino-5-oxonorleucine. The number of the cross-links per molecule was significantly lower than might be expected in order to fully stabilize the molecule. These results suggest that the keto-imine cross-links in type IV collagen have been stabilized in part by conversion into an unknown non-reducible form, although a sensitive immunoassay failed to show the presence of any pyridinoline. Comparison with the fibrous collagen from placenta suggested that the rate of this conversion is much greater in basement-membrane collagen.
D001485:Basement Membrane / Q000032:analysis; D001894:Borohydrides; D002852:Chromatography, Ion Exchange; D003094:Collagen; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008854:Microscopy, Electron; D008958:Models, Molecular; D010434:Pepsin A; D010446:Peptide Fragments / Q000032:analysis; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D011487:Protein Conformation; D013997:Time Factors
D001894:Borohydrides; D010446:Peptide Fragments; C025364:sodium borohydride; D003094:Collagen; D010434:Pepsin A