The biosynthesis of basement-membrane collagen by isolated rat glomeruli.
A technique is described for the rapid isolation of highly purified preparations of viable glomeruli from rat kidney cortex. The synthesis of protein as judged by the incorporation of [14C]proline into non-diffusible material was shown to be linear for up to 6 h. The synthesis of collagen, measured as non-diffusible 4-hydroxy[14C]proline, was also linear over this period but represented only a small proportion of total protein synthesis. Similar studies conducted in vivo confirmed that collagen synthesis accounted for less than 5% of total protein synthesis in glomeruli. When isolated glomeruli were incubated with [14C]proline, it was found that approximately 16% of the hydroxyproline present in the collagenous component occurred as the 3-isomer. When glomeruli were incubated with [14C]lysine over 90% of the hydroxy[14C]lysine synthesised was glycosylated and most of the glycosylated hydroxy[14C]lysine was present as glucosyl-galactosyl-hydroxy[14C]lysine. The size of the basement membrane collagen synthesised by the isolated glomeruli was estimated by treating the 14C-labelled protein with mercaptoethanol and sodium dodecyl sulphate and then chromatographing the 14C-labelled protein on an agarose column equilibrated and eluted with buffer containing 0.1% (w/v) sodium dodecyl sulphate. The initial form of [14C]collagen synthesised was found to consist of polypeptide chains which had molecular weights of approximately 140 000 and which were shown to be distinctly larger than the polypeptide chains from embryonic chick tendon procollagen. Also when glomeruli were labelled with [14C]proline for 2 h and chased with unlabelled proline for 4 h there was a time-dependent conversion of the initially synthesised collagen moiety to collagen polypeptide chains which co-chromatograph with tendon pro-alpha chains (molecular weight approx. 120 000).
D000818:Animals; D001485:Basement Membrane / Q000166:cytology / Q000378:metabolism*; D001835:Body Weight; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D003094:Collagen / Q000096:biosynthesis*; D006901:Hydroxylysine / Q000096:biosynthesis; D006909:Hydroxyproline / Q000096:biosynthesis; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000378:metabolism*; D007678:Kidney Glomerulus / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism; D008297:Male; D011392:Proline / Q000378:metabolism; D051381:Rats; D013997:Time Factors
D006901:Hydroxylysine; D003094:Collagen; D011392:Proline; D006909:Hydroxyproline