Structural glycoprotein from the media of pig aorta. Aggregation of the S-carboxamidomethyl subunits

Meida of pig aorta was extracted with 1 M NaCl and 2 M MgCl 2 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 (∼ 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 [46]. Des expériences sont décrites en vue d'isoler et de caractériser les glycoprotéines associées au stroma fibreux insoluble de la média de l'aorte par des méthodes aussi douces que possible. Une fraction glycoprotidique a pu être isolée à partir de la média de l'aorte de porc possédant les caractéristiques des glycoprotéines de structure obtenues précédemment par des méthodes plus dégradantes [5]. Le collagène, les protéoglycannes et les glycoprotéines solubles de la média aortique ont été extraits par NaCl 1 M et MgCl 2 2 M. Les glycoprotéines restant dans le résidu ont été extraites par l'urée 6 M-mercaptoéthanol 0,1 M. Les protéines extraites par l'urée ont été précipitées par dialyse, redissoutes dans la guanidine HCl 4M-DTT 0,05 M et ont été S-carb