Newly Synthesized Proteins in Rat Glomerular Fractions

Abstract

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 a 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.

Glycoprotein Synthesis of Renal Collecting Duct Epithelium Cultured as Globular Bodies

“…Only a third of the acidic 85,000 dalton glycopro tein was recovered in the second-dimen sional electrophoresis: the rest was hydro lyzed by the low pH during the isoelectrofocusing procedure. A similar acid-and pHsensitive glomerular glycoprotein has been described earlier [4],…”

“…Recently, however, cultures of glomerular [1] and tubular cells [2,3] have been devel oped but most often have been used to mea sure either transport capabilities or hor monal responses. Only in relatively few cases have radiolabellcd precursor incuba tions been performed to study the synthesis of glycoproteins of whole glomeruli [4] and the glycosaminoglycan biosynthesis of epi thelial and mesangial cells [5]. Studies con cerning membrane glycoproteins and cell surface proteins were undertaken in BHK cells [6] and MDCK cells [7], both undefined renal cell lines.…”