Newly synthesized proteins in rat glomerular fractions.
Isolated glomerular preparations are known for their capability to synthesize proteins. A thorough biochemical separation and characterization of newly synthesized proteins, however, is lacking. In this study isolated glomeruli from rat kidney (enriched by sieving technique alone or by a combination of sieving and subsequent further enrichment by either sucrose or Ficoll discontinuous centrifugation) were incubated for various periods with protein and glycoprotein precursors. The synthesis of soluble and membrane-bound proteins was studied. Separation of glomerular proteins was carried out by means of SDS-electrophoresis in 6 M urea. The radioactivity of newly synthesized proteins was determined by the scintillation technique. In the leucine-incubated glomeruli, a broad spectrum of newly synthesized monomer proteins ranging from 90,000 to 25,000 daltons was detected, whereas in the glucosamine and galactose incubated glomeruli synthesis of glycoproteins of molecular weights ranging mainly between 90,000 and 60,000 daltons could be observed. Most of the radioactivity incorporated into isolated glomeruli was regained in 600 g (containing basement membrane with residual mesangial/endothelial nuclei and cellular debris) and 12,000 g (membrane structures and mitochondria) pellets, indicating that most of the newly synthesized proteins are membrane bound. A component of newly synthesized glycoprotein (72,000 daltons) could be extracted by mild NaCl treatment and is partly soluble in desoxycholate. Further experiments utilizing isoelectrofocusing and two-dimensional electrophoresis are in progress.
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005690:Galactose / Q000494:pharmacology; D005944:Glucosamine / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D066298:In Vitro Techniques; D007678:Kidney Glomerulus / Q000378:metabolism* / Q000648:ultrastructure; D007930:Leucine / Q000494:pharmacology; D008565:Membrane Proteins; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains
D006023:Glycoproteins; D008565:Membrane Proteins; D011506:Proteins; D007930:Leucine; D005944:Glucosamine; D005690:Galactose