Biosynthesis of rabbit serum albumin in a heterologous fractionated subcellular system.
As demonstrated by a simple procedure based on indirect immunoprecipitation, proteins retained on heparin-Sepharose 4B from postmitochondrial supernatants of rat liver and Zajdela hepatoma catalyse the translation of rabbit serum albumin mRNA in the presence of ribosomal subunits from rat liver, Zajdela hepatoma or rabbit reticulocytes. The albumin synthesis shows an optimum at 1.5 mM MgCl2 and 25 mM KCl and requires ATP and GTP. It is significantly stimulated by tRNA and proceeds for more than 2 h, suggesting a high rate of reinitiation. At the optimum ribosomes:mRNA ratio of 13:1, the immunoprecipitable radioactivity exceeds 15-20-times the blank values. Fluorography of polyacrylamide slabs after electrophoresis of immunoprecipitates revealed the presence of only complete full-size serum albumin without any smaller peptides resulting from premature terminations of polypeptide chains, demonstrating faithful translation. In stained gels only, both heavy and light chains of immunoglobulin G were found, indicating that the assay procedure is highly specific and reliable. The fractionated heterologous protein-synthesizing system described in this paper may be generally useful for studies on the synthesis of specific proteins and factors affecting their rates since, unlike comparable translation assays, a precise calculation of the balance of newly synthesized proteins is possible.
D000818:Animals; D007120:Immunochemistry; D066298:In Vitro Techniques; D008099:Liver / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012709:Serum Albumin / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000378:metabolism
D012709:Serum Albumin