title: Macromolecular organization of basement membranes. Characterization and comparison of glomerular basement membrane and lens capsule components by immunochemical and lectin affinity procedures.
abstract: The macromolecular components of bovine glomerular basement membrane (GBM) and lens capsules (anterior and posterior) solubilized by sequential extractions with denaturing agents were quantitated and characterized by polyacrylamide gel electrophoresis, CL-6B filtration, and DEAE-cellulose chromatography with the help of immunochemical techniques. Laminin, entactin, fibronectin, and heparan sulfate proteoglycan were primarily recovered (over 80%) from both basement membranes in a guanidine HCl extract which contained only a limited amount of the total protein (10-14%); most of the remainder of these noncollagenous components could be solubilized by the guanidine in the presence of reducing agent. Although a portion of the Type IV collagen could be obtained by these treatments, effective extraction of this protein depended on exposure to sodium dodecyl sulfate under reducing conditions. Immunoblot analysis revealed a remarkably similar pattern for GBM and lens capsule Type IV collagens with prominent bands of Mr = 390,000, 210,000, and 190,000 being evident. Fibronectin was present in much greater amounts in GBM than lens capsule while the reverse was true for entactin. In both GBM and lens capsules, the entactin (Mr = 150,000) exceeded laminin; the latter protein on immunoblotting was found to contain primarily the alpha-subunit (Mr = 200,000). The size of the heparan sulfate proteoglycan from anterior (Mr = 400,000) and posterior lens capsule (Mr greater than 500,000) was substantially larger than that from GBM (Mr = 200,000). During DEAE-cellulose chromatography under nonreducing conditions in a denaturing solvent, a portion of the Type IV collagen coeluted with the proteoglycan from these membranes. Considerable Bandeiraea simplicifolia I binding activity (alpha-D-galactose specific) was observed in GBM and lens capsule extracts and column fractions which could not be accounted for by laminin alone. Several components which reacted with this lectin were seen on transblots and among these Type IV collagen was identified. In contrast to the basement membranes from bovine tissues, the constituents from human GBM did not react with the B. simplicifolia I lectin.
mesh_terms: D000818:Animals; D001485:Basement Membrane / Q000648:ultrastructure*; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000032:analysis; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003094:Collagen / Q000032:analysis; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005353:Fibronectins / Q000032:analysis; D006023:Glycoproteins / Q000032:analysis; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000032:analysis; D006801:Humans; D007158:Immunologic Techniques; D007678:Kidney Glomerulus / Q000648:ultrastructure*; D007797:Laminin / Q000032:analysis; D037102:Lectins / Q000378:metabolism*; D007908:Lens, Crystalline / Q000032:analysis*; D008562:Membrane Glycoproteins; D008970:Molecular Weight; D012995:Solubility
chemical_list: D011508:Chondroitin Sulfate Proteoglycans; D003094:Collagen; D004229:Dithiothreitol; D005353:Fibronectins; D006023:Glycoproteins; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D007797:Laminin; D037102:Lectins; D008562:Membrane Glycoproteins; C029967:nidogen