Cell-free translation and thyroxine induction of carbamyl phosphate synthetase I messenger RNA in tadpole liver.
Total RNA of tadpole and frog (Rana catesbeiana) liver was isolated by either 7 or 8 M guanidine . HCl extraction and translated in a cell-free protein-synthesizing system derived from rabbit reticulocytes. The identity of carbamyl phosphate synthetase I[carbamoyl-phosphate synthase (ammonia); ATP:carbamate phosphotransferase (dephosphorylating), EC 2.7.2.5] synthesized in vitro with the purified enzyme was established as follows: (i) immunoprecipitation by a specific antibody; (i) comigration with purified carrier enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis; (iii) copurification with carrier enzyme by affinity chromatography on Cibacron Blue F3GA-coupled agarose; and (iv) formation of identical proteolytic cleavage products. Inclusion of protease inhibitors in the system resulted in no apparent change in the polypeptide molecular weight. These results indicate that carbamyl phosphate synthetase I is synthesized as a polypeptide that is indistinguishable from the mature enzyme by the analytical methods used and that it is not grossly modified during its transport into mitochondria. The level of translatable mRNA for carbamyl phosphate synthetase-I in tadpole liver was increased about 2-fold 1 day after thyroxine treatment and did not change significantly through 4 subsequent days of treatment. Thus the thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver is at least partly due to an increase of translatable mRNA for this enzyme.
D000818:Animals; D001001:Anura; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000096:biosynthesis*; D002474:Cell-Free System; D004792:Enzyme Precursors / Q000096:biosynthesis*; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism*; D010770:Phosphotransferases / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000096:biosynthesis*; D011892:Rana catesbeiana; D013974:Thyroxine / Q000494:pharmacology*
D004792:Enzyme Precursors; D012333:RNA, Messenger; D012313:RNA; D010770:Phosphotransferases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D013974:Thyroxine