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The synthesis and degradation of the renal glomerular basement membrane have been investigated in the rat with the aid of injected tracer doses of various tritiated amino acids including L-proline, L-lysine, L-phenylalanine, L-leucine, and glycine. After incorporation into the basement membrane the turnover times of these amino acid constituents, as well as of hydroxyproline and hydroxylysine, were determined from the decay in their specific radioactivities. The loss of radioactivity from the proline and hydroxyproline of the glomerular basement membrane was as slow as that from tail tendon collagen in the same animals (turnover time of more than 100 days) and contrasted with the radiodecay of the proline in other glomerular proteins (turnover time of 9 days). The glycine of the membrane similarly turned over at this very slow rate. The total replacement times of the leucine, hydroxylysine, lysine, and phenylalanine constituents of the basement membrane were determined to be somewhat shorter with a range of 65 to 23 days. The nonuniform turnover of these membrane components may be a function of the polydispersity of the peptide subunits of the basement membrane observed by polyacrylamide gel electrophoresis and may reflect a subtle morphological and functional heterogeneity. The amino acid and saccharide composition of the rat glomerular basement membrane used in these studies are also reported.

Three major glycoproteins of calf thyroid plasma membranes were preferentially solubilized by chloroform/methanol extraction and recovered along with glycolipids in the aqueous phase. After removal of lipid from this extract, a fraction was obtained which accounted for about 20% of the carbohydrate of the membrane but only 2% of its peptide weight. Partial resolution of the components could be achieved by filtration on Bio-Gel A-5m, while preparative polyacrylamide gel electrophoresis resulted in the isolation in homogeneous form of approximately equal amounts of the three glycoproteins which were designated as GP-1, GP-2, and GP-3, in order of their increasing mobility. These purified glycoproteins appeared on electrophoresis as single components by periodic acid-Schiff staining as well as by distribution of radioactivity following 3H or 14C labeling. Molecular weights of 100,000, 59,000, and 20,000 were estimated for the three components on the basis of their retardation coefficients. The total carbohydrate content by weight determined for GP-1, GP-2, and GP-3 was 56, 57, and 79%, respectively. The sugar constituents were mannose, galactose, fucose, glucosamine, galactosamine, and sialic acid, which were present in the following mol per cents: GP-1, 13:32:5:24:13:12; GP-2, 20:28:3:32:5:11; GP-3, 12:36:2:34:6:10. Studies performed with various lectins (Bandeiraea simplicifolia I and I (B4), wheat germ, Ricinus communis, and soybean) on the gycoproteins, either native or after treatment with glycosidases (alpha- or beta-galactosidase, neuraminidase), indicated that sialic acid and alpha-linked galactose were in terminal positions, beta-galactosyl residues were internally located, and chains containing the sequence sialic acid-N-acetylgalactosamine were present.