Open AccessTranslation of mRNA from Rat‐Liver Polysomes into Tyrosine Aminotransferase and Tryptophan Oxygenase in a Protein‐Synthesizing System from Wheat Germ
Author(s) -
ROEWEKAMP Walter G.,
HOFER Erhard,
SEKERIS Constantin E.
Publication year - 1976
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1976.tb10977.x
Subject(s) - tyrosine aminotransferase , polysome , messenger rna , biochemistry , biology , protein biosynthesis , enzyme , chemistry , microbiology and biotechnology , enzyme inducer , rna , ribosome , gene
Messenger RNA was isolated from rat liver polysomes by phenol chloroform extraction and subsequent oligo(dT)‐cellulose chromatography. The mRNA was translated in a protein‐synthesizing system in vitro derived from wheat germ. The system was optimized in respect to Mg 2+ and K + . The presence of spermidine or spermine is necessary for the synthesis of polypeptides having molecular weights of over 20000. In the absence of the bases only small molecular weight products are formed. The amount of protein synthesized is linearly dependent on the amount of mRNA added up to concentrations of 80 μg mRNA/ml. The synthesis of tyrosine aminotransferase and tryptophan oxygenase it the system in vitro has been demonstrated by specific immunoprecipitation and sodiumdodecylsulfate polyacrylamide gel electrophoresis of the precipitate with enzyme proteins as marker. The amount of specific product formed is linearly dependent on the amount of mRNA present. The amount of translatable tyrosine aminotransferase mRNA and tryptophan oxygenase mRNA increases after administration of hydrocortisone to adrenalectomized rats. At low doses of hormone (2 mg 100 g body weight) maximal values are observed at 4 h, control levels being reached at 6–8 h after hormone application. With higher doses of hydrocortisone (20 mg/100 g body weight) maximal values are attained at 6 h, tending to control levels 14 h after treatment. The enzyme activity curves are parallel to the mRNA curves, the peak of enzyme activity occurring 2 h after the peak of mRNA activity.