BIOSYNTHESIS OF BASEMENT-MEMBRANE COLLAGEN IN CULTURES OF RENAL GLOMERULAR AND TUBULAR EPITHELIAL-CELLS

  • 1 January 1975
    • journal article
    • research article
    • Vol. 1  (4) , 227-234
Abstract
Confluent cultures of [human] renal glomerular or tubular epithelial cells were incubated with [14C]-proline and [3H]-lysine. The incorporation rate of both radioactive precursors was linear for up to 12 h. The synthesis and secretion of basement membrane collagenous polypeptides was demonstrated by the presence in the culture media of non-dialyzable 4-hydroxy [14C]-proline and hydroxy [3H]-lysine. After gel filtration of the culture media on Sephadex G-100 columns, glomerular and tubular basement membrane polypeptides were identified in the chromatographic fractions by radioimmunoassay. They were further purified by affinity chromatography, using Sepharose CNBr coupled with specific rabbit anti-human glomerular or tubular basement membrane antibodies. Adsorbed labeled membrane polypeptides were eluted from the Sepharose by acidic medium at 4.degree. C. This membrane material represented 3-4% of the total proteins synthesized by glomerular and tubular cells. The glomerular and tubular basement membrane polypeptides purified by affinity chromatography exhibited a MW of approximately 140,000; 80% of the total hydroxy [3H]-lysine was recovered as glucosyl-galactosyl-hydroxy [3H]-lysine. Analysis of the carbohydrate content of labeled basement membrane polypeptide chains originating from glomerular or tubular cells incubated with [14C]-glucose or [14C]-glucosamine indicated the presence of glucose, galactose, mannose, glucosamine and galactosamine. No fucose, mannosamine or sialic acid were detectable. Glomerular and tubular epithelial cells are able to synthesize basement membrane collagenous polypeptides in culture. This property might provide a useful tool for the study of the biosynthesis of similar material by diseased kidneys.