Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits.
Media of pig aorta was extracted with 1 M NaCl and 2 M MgCl2 to remove most of the soluble collagen, proteoglycans and glycoproteins. The glycoproteins remaining in the residue were extracted with 6 M urea-0.1 M mercaptoethanol. The urea soluble proteins were precipitated by dialysis, redissolved in 4 M guanidine-0.05 M DTT and were S-carboxamidomethylated (CM-guanidine extract). This extract was further fractionated by a variety of methods in order to separate a glycoprotein from collagen and proteoglycans. Caesium chloride density-gradient ultracentrifugation of the CM-guanidine extract separated a minor proteoglycan peak from a major glycoprotein fraction still containing some hydroxyproline. This major glycoprotein fraction was excluded as a single peak from Sephadex G 100 and G 200 in 4 M guanidinium chloride or in 6 M urea-0.2 per cent SDS. Sodium dodecylsulphate gel electrophoresis separated this high molecular weight Sephadex fraction into a major low molecular weight (approximately 35000 daltons) component and a minor high molecular weight component. This glycoprotein fraction could also be separated from a collagenous fraction and from proteoglycans by ion exchange chromatography on DEAE cellulose or by gelfiltration on Sepharose 4 B in 6 M urea-0.02 M EDTA-0.2 per cent SDS at pH 7.0. The isolated glycoprotein fraction is rich in dicarboxylic amino acids, contains galactose, mannose, (glucose), N-acetylglucosamine and sialic acid. The S-carboxamidomethyl glycoprotein preparation interacts with acid soluble calf skin collagen on isoelectric focusing in sucrose gradient in urea. This interaction is in favour of the biological role claimed for structural glycoproteins during fibrogenesis and differentiation.
D000596:Amino Acids; D000818:Animals; D001013:Aorta, Thoracic / Q000032:analysis*; D055598:Chemical Phenomena; D002621:Chemistry; D006020:Glycopeptides; D006023:Glycoproteins / Q000302:isolation & purification; D006595:Hexosamines; D006603:Hexuronic Acids; D006863:Hydrogen-Ion Concentration; D006909:Hydroxyproline; D046911:Macromolecular Substances; D003012:Microbial Collagenase; D012995:Solubility; D013552:Swine
D000596:Amino Acids; D006020:Glycopeptides; D006023:Glycoproteins; D006595:Hexosamines; D006603:Hexuronic Acids; D046911:Macromolecular Substances; D003012:Microbial Collagenase; D006909:Hydroxyproline