Biosynthesis of basement membrane matrix by isolated rat renal glomeruli.

Biosynthesis of basement membrane matrix by isolated rat renal glomeruli. The incorporation of radioactive precursors into the extracellular basement membrane matrix has been investigated in a purified preparation of isolated rat kidney glomeruli. Using deoxycholate extraction of isolated glomeruli which were incubated with radioactively labeled amino acids and carbohydrates, as an assay system for the measurement of incorporation of basement membrane precursors into intact deoxycholate insoluble basement membrane material, we demonstrated the in vitro biosynthesis of this structure. The assay system minimized the possibility of contamination of the isolated labeled basement membrane with labeled cell membrane fragments which may occur with the standard isolation procedures involving sonication and centrifugation. A linear incorporation of labeled proline, lysine, glycine, glucosamine, and galactose into glomerular basement membrane was shown. Basement membrane synthesis was inhibited by metabolic poisons and protein synthesis inhibitors as well as by inhibitors of collagen synthesis, but not by colchicine, an inhibitor of collagen secretion. The appearance of 14 C-hydroxyproline in the basement membrane matrix was negligible during the first 4 hours of incubation and rose only to 1% of the total proline counts thereafter. The results are consistent with the characterization of glomerular basement membrane synthesis and deposition as a two component system comprising a rapidly synthesized and deposited noncollagenous glycoprotein component(s) and a collagenous component which is only deposited after a delay of 4 to 6 hours. Biosynthese de la matrice de la membrane basale par le glomerule isole de rat. L'incorporation de precurseurs radioactifs dans la matrice de la membrane basale extracellulaire a ete etudiee dans une preparation purifiee de glomerules isoles de rat. L'utilisation d'extrait par le deoxycholate de glomerules isoles incubes avec des acides amines et des hydrates de carbone marques comme systeme de mesure de l'incorporation des precurseurs de la membrane basale par la fraction insoluble dans le deoxycholate de la membrane basale a permis de demontrer la biosynthese in vitro de cette structure. Le systeme de mesure minimise la contamination possible de la membrane basale isolee marquee par des fragments de membrane cellulaire marques qui peuvent exister du fait des procedes courants d'isolement qui impliquent les ultra-sons et la centrifugation. Une incorporeation lineaire de proline, lysine, glycine, glucosamine, et galactose marques a ete mise en evidence dans la membrane basale glomerulaire. La synthese de membrane basale est inhibee par les poisons metaboliques et les inhibiteurs de la synthese proteique mais pas par la colchicine, un inhibiteur de la secretion de collagene. L'apparition de 14 C-hydroxyproline dans la matrice de la membrane basale est negligeable durant les 4 premieres heures d'incubation et n'atteint que 1% de la proline totale ensuite. Les resultats sont en faveur de la definition de la synthese et de la deposition de membrane basale glomerulaire comme un systeme a deux composants incluant un ou des composants glycoproteine(s) non collagenes rapidement synthetises et deposes et un composant collagene qui est depose seulement apres un delai de 4 a 6 heures.