Synthesis Of Carbamyl Phosphate Synthetase Research Articles

Biogenesis of the mitochondrial enzyme, carbamyl phosphate synthetase Appearance during fetal development of rat liver and rapid repression in freshly dispersed hepatocytes

Synthesis of carbamyl phosphate synthetase was undetectable in fetal rat liver at 16 days gestation but by 4–5 days after birth (11–12 days later), this single protein accounted for approx. 5% of total liver protein and roughly 1% of total liver protein synthesis. Likewise, translatable mRNA coding for the enzyme was absent from 16-day fetal livers but then rapidly accumulated reaching maximum levels just after birth. The in vitro primary translation product of carbamyl phosphate synthetase mRNA corresponded to a higher molecular weight biosynthetic precursor of the enzyme; peptide maps obtained from the precursor synthesized both in vivo and in vitro and from the mature enzyme made in vivo were the same. When livers of neonatal rats were perfused with collagenase and further treated to yield a preparation of freshly dispersed hepatocytes highly active in general protein synthesis, a procedure which took about 45 min to complete, biosynthesis of carbamyl phosphate synthetase was found to be completely absent in these cells. The mRNA coding for the enzyme, however, could be extracted from the dispersed hepatocytes and was actively translatable in vitro, at levels approximately 75% of those for mRNA obtained from intact liver. Repression of biogenesis of carbamyl phosphate synthetase in dispersed hepatocytes, therefore, must involve a mechanism which shifts the mRNA coding for the enzyme out of active polysomal complexes and renders it further untranslatable in vivo but not in vitro.

Chloramphenicol inhibits hormone-dependent induction of cytoplasmic mRNA coding for the mitochondrial enzyme, carbamyl phosphate synthetase, in Rana catesbeiana tadpoles

In tadpoles injected with thyroxine (T 4), synthesis of carbamyl phosphate synthetase 1 is induced so that within 7–8 days this single polypeptide represents one of the most abundant proteins in the liver. Translational assays in vitro showed that liver RNA from control animals had very low levels of translatable mRNA coding for the enzyme whereas carbamyl phosphate synthetase mRNA activity was prominent in liver from tadpoles which had been treated with T 4 for just 2 days. When the primary translation product of carbamyl phosphate synthetase mRNA was immunoprecipitated from a messenger-dependent rabbit reticulocyte cell-free system programmed with total liver mRNA, it migrated on SDS-polyacrylamide gels more slowly than the in vivo form of the enzyme and otherwise demonstrated characteristics which were very similar to the precursor for carbamyl phosphate synthetase previously described in rat liver. If tadpoles were treated for 2 days with T 4 plus an inhibitor of mitochondrial protein synthesis, chloramphenicol, T 4-dependent induction of both enzyme synthesis and translatable carbamyl phosphate synthetase mRNA activity were repressed by 45–65%. The two measurements, synthesis in vivo and mRNA activity in vitro, were made on the same liver and correlated closely in their response to chloramphenicol. The data suggest that a product of mitochondrial protein synthesis may be involved in mediating hormonal regulation of the nuclear gene coding for carbamyl phosphate synthetase.

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.

Antipain inhibits thyroxine-induced synthesis of carbamyl phosphate synthetase I in tadpole liver.

The increased activity of carbamyl phosphate synthetase I [carbamoyl-phosphate synthase (ammonia); ATP: carbamate phosphotransferase (diphosphorylating), EC 2.7.2.5] in tadpole liver observed during thyroxine-induced metamorphosis was markedly inhibited by intraperitoneal injection of the microbial protease inhibitor antipain (0.1 micrometermol/g of body weight, twice daily). A somewhat less than maximal inhibition was seen when antipain was given only during the first 2 days of thyroxine treatment. On the other hand, little inhibition was observed when the inhibitor was given after the third or fourth day of thyroxine treatment. Antipain also inhibited thyroxine-induced increases of ornithine transcarbamylase (EC 2.1.3.3), arginase (EC 3.5.3.1), and succinate-cytochrome c reductase (EC 1.3.99.1) activities. Among other microbial protease inhibitors tested, chymostatin was nearly as effective as antipain, leupeptin was less effective, and pepstatin was ineffective. Analysis of the total liver protein and of the immunoprecipitate by sodium dodecyl sulfate/polyacrylamide gel electrophoresis showed that the inhibition was due to decreased amount of the enzyme protein. Antipain had no significant effect on leucine incorporation into total protein of tadpole liver. These results indicate the involvement of a proteolytic step in the pretranscriptional events in thyroxine-stimulated enzyme induction.

Isolation and partial characterization of an argR mutant of Salmonella typhimurium

An arginine regulatory mutant (i.e., mutated in the argR gene) has been isolated from a strain of Salmonella typhimurium LT2. The argR mutant was found to excrete arginine into the growth medium with glycerol but not glucose as carbon source. Constitutive synthesis of arginine biosynthetic enzymes was observed. Whereas previous results (A. T. Abd-E1-A1 and J. L. Ingraham, Abstr. Annu. Meet. Am. Soc. Microbiol. 1975, K169, p. 175) have shown constitutive synthesis of carbamyl phosphate synthetase in the argR mutant, the regulation of the synthesis of the last five enzymes of the pyrimidine pathway was unaffected. However, in pyrH mutants, known to exhibit derepressed synthesis of the pyrimidine enzymes, a 10-fold derepression of ornithine transcarbamylase was observed.

Control of Carbamyl Phosphate Synthesis in Salmonella typhimurium

Abstract Carbamyl phosphate synthetase from Salmonella typhimurium LT-2 has been purified 320-fold; the modulation of enzyme activity by substrate concentration, effectors, and the effect of temperature on this modulation have been studied. Uridine phosphate inhibits the enzymic reaction; ornithine reverses this inhibition and also activates the enzyme in the absence of UMP by decreasing the apparent Km for ATP. The stimulation by ornithine is greater at low temperature, whereas the inhibitory effect of UMP is equal at high and low temperatures. Studies with various strains blocked by mutation in steps which interconvert cytosineand uracil-containing compounds have established that the pyrimidine repressor of the synthesis of carbamyl phosphate synthetase is a cytosine-containing compound.

Actinomycin D inhibition of thyroxine-induced synthesis of carbamyl phosphate synthetase

Actinomycin D inhibition of thyroxine-induced synthesis of carbamyl phosphate synthetase

Synthesis of Carbamyl Phosphate Synthetase in Liver Slices From Thyroxine-treated Tadpoles

Synthesis of Carbamyl Phosphate Synthetase in Liver Slices From Thyroxine-treated Tadpoles

The Synthesis of Carbamyl Phosphate Synthetase in Thyroxin-treated Tadpoles

The Synthesis of Carbamyl Phosphate Synthetase in Thyroxin-treated Tadpoles

Biochemical studies on amphibian metamorphosis. I. The effect of thyroxine on protein synthesis in the tadpole.

Thyroxine has been shown to accelerate the synthesis of carbamyl phosphate synthetase in the liver of Rana catesbeiana. Stimulation of carbamyl phosphate synthetase synthesis by thyroxine appears to be relatively specific because of the following observations: (1) succinoxidase activity decreased during the time that carbamyl phosphate synthetase increased; (2) liver catalase responded more slowly than carbamyl phosphate synthetase to thyroxine; (3) the ratio of biochemical changes/morphological changes was greatly altered during thyroxine-induced metamorphosis. The relationships between the concentration of thyroxine and (1) temperature; (2) duration of exposure of the tadpole to thyroxine; and (3) the activity of carbamyl phosphate synthetase during the induced synthesis of carbamyl phosphate synthetase by thyroxine are discussed. Chloramphenicol and thiouracil partly counteracted the effect of thyroxine on the synthesis of carbamyl phosphate synthetase.