Arterial basement-membrane-like material isolated and characterized from rabbit aortic myomedial cells in culture.
Arterial basement-membrane-like material was isolated from rabbit aortic myomedial cell cultures by sonication and differential centrifugation. Isolated basement-membrane-like material was shown to be free of both cellular and matrix contaminants, on the basis of determinations of DNA, RNA, cholesterol, phosphorus and (Na+ + K+)-activated ATPase, combined with electron microscopy. Amino acid analyses showed that arterial basement-membrane-like material was composed of predominantly non-collagenous amino acids. Evaluated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, reduced basement-membrane-like material comprised six major and about 30 minor components in the Mr range 10 000-600 000. One of the major peptides (Mr 225 000) was disulphide-linked. Periodic acid-Schiff staining of gels indicated that most high-molecular-weight components were glycoproteins. Two-dimensional gel electrophoresis resolved reduced basement-membrane-like material into more than 100 components, with pI from 5 to 7. The disulphide-linked Mr-225 000 peptide appeared heterogeneous, with pI of 5.6-6.0, and was considered to represent fibronectin. All major peptides were of non-collagenous nature, on the basis of their susceptibility to pepsin and resistance to collagenase. Purified myomedial basement-membrane-like material contained collagenous peptides, as indicated by the presence of hydroxyproline and hydroxylysine. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of pepsin-treated and reduced basement-membrane-like material revealed five high-molecular-weight collagenous components appearing in the Mr range 105 000-375 000 relative to type I collagen standards.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001013:Aorta, Thoracic / Q000166:cytology / Q000378:metabolism*; D001485:Basement Membrane / Q000166:cytology / Q000378:metabolism; D002469:Cell Separation; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013010:Sonication
D000596:Amino Acids; D010446:Peptide Fragments