Carbohydrate and Amino‐Acid Composition of Human Glomerular‐Basement‐Membrane Fractions Purified by Affinity Chromatography

Human glomerular basement membranes can be substantially solubilized by autoclaving in 0.1 M Tris‐acetate buffer pH 7.4, at 110°C, during 3 h or by treatment with 8 M urea containing mercaptoethanol. The best solubilization (80% of soluble material) was obtained by autoclaving. Therefore it was this material which has been purified from tritiated glycoproteins, by affinity chromatography, using Sepharose cyanogen‐bromide coupled with rabbit anti‐human glomerular‐basement‐membrane γ‐G globulin; at least 80% of solubilized basement membranes are adsorbed onto this γ‐globulin. Unadsorbed material contains the same carbohydrate units as the whole membranes but are richer in heteropolysaccharide units. Its molecular weight is lower than 69000. Adsorbed glyco‐protein components, eluted from the Sepharose by acidic medium at 4°C, are further purified by preparative polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. Four major fractions exhibiting molecular weights from 80000 to 300000 are obtained; they have the same carbohydrate units as the whole membranes, but have more disaccharide units. Similarly, amino acid composition of these purified fractions exhibits more 3‐hydroxyproline and hydroxy‐lysine residues than the whole membranes. This procedure appears therefore to be a valuable technique for preparing large quantities of labelled glomerular‐basement‐membrane antigens containing both types of oligosaccharidic chains.