Purification and structural characterization of intact and fragmented nidogen obtained from a tumor basement membrane.
Extraction of a basement-membrane-producing mouse tumor with 6 M guanidine/HCl in the presence of protease inhibitors allowed the purification of the genuine form of the matrix protein nidogen (Mr = 150,000) and, in addition, two defined fragments (Mr = 130,000 and 100,000). Smaller fragments (Mr = 80,000 and 40,000) were obtained under conditions with less stringent control of endogenous proteolysis. Intact nidogen and the larger fragments were similar in amino acid and carbohydrate (about 5%) composition, the presence of a single polypeptide chain, conformational features as revealed by CD spectroscopy and all shared major epitopes located on the Mr = 80,000 fragment. Additional epitopes were found on intact nidogen and the Mr = 130,000 fragment. Nidogen and the various fragments possess different N-terminal amino acid sequences indicating a stepwise degradation from the N-terminal end of the molecule. Electron microscopical and hydrodynamic studies of the Mr = 80,000 fragment demonstrated a structure consisting of a globular head connected to a thin tail. Intact nidogen appears to contain a somewhat larger globule but the same tail, which is terminated at its opposite end by a second, smaller globular structure. The data suggest a multidomain structure for nidogen containing sites highly susceptible to proteolytic cleavage.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001485:Basement Membrane / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D002845:Chromatography / Q000379:methods; D002848:Chromatography, DEAE-Cellulose; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D007120:Immunochemistry; D007526:Isoelectric Point; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000302:isolation & purification*; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D009374:Neoplasms, Experimental / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification*; D011487:Protein Conformation; D014461:Ultracentrifugation
D000596:Amino Acids; D002241:Carbohydrates; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D010446:Peptide Fragments; C029967:nidogen