The influence of ammonia on purine and pyrimidine nucleotide biosynthesis in rat liver and brain in vitro

1. The effect of ammonia on purine and pyrimidine nucleotide biosynthesis was studied in rat liver and brain in vitro. The incorporation of NaH¹⁴CO₃ into acid-soluble uridine nucleotide (UMP) in liver homogenates and minces was increased 2.5–4-fold on incubation with 10mm-NH₄Cl plus N-acetyl-l-glutamate, but not with either compound alone. 2. The incorporation of NaH¹⁴CO₃ into orotic acid was increased 3–4-fold in liver homogenate with NH₄Cl plus acetylglutamate. 3. The 5-phosphoribosyl 1-pyrophosphate content of liver homogenate was decreased by 50% after incubation for 10min with 10mm-NH₄Cl plus acetylglutamate. 4. Concomitant with this decrease in free phosphoribosyl pyrophosphate was a 40–50% decrease in the rates of purine nucleotide synthesis, both de novo and from the preformed base. 5. Subcellular fractionation of liver indicated that the effects of NH₄Cl plus acetylglutamate on pyrimidine and purine biosynthesis required a mitochondrial fraction. This effect of NH₄Cl plus acetylglutamate could be duplicated in a mitochondria-free liver fraction with carbamoyl phosphate. 6. A similar series of experiments carried out with rat brain demonstrated a significant, though considerably smaller, effect on UMP synthesis de novo and purine base reutilization. 7. These data indicate that excessive amounts of ammonia may interfere with purine nucleotide biosynthesis by stimulating production of carbamoyl phosphate through the mitochondrial synthetase, with the excess carbamoyl phosphate in turn increasing pyrimidine nucleotide synthesis de novo and diminishing the phosphoribosyl pyrophosphate available for purine biosynthesis.     

Effect of thyroid hormone on peroxisomal flavin enzymes in rat liver and kidney

The effect of thyroid hormone on peroxisomal enzyme activity was studied in thyroidectomized- and T4-administered-thyroidectomized rats. In liver, the activities of isozyme A of L-alpha-hydroxyacid oxidase, D-amino acid oxidase, urate oxidase and catalase were decreased by thyroidectomy, and the diminished enzyme activities were restored by T4 administration to rats. These modifications induced by thyroidectomy or by T4 administration, however, were prominent only in immature animals (20-day-old rats). Although the changes in-alpha-hydroxyacid oxidase and D-amino acid oxidase activities, induced by thyroidectomy or by T4 administration, were also observed in 40-day-old rats, those in urate oxidase and catalase activities were not significant in 40-day-old rats. Acyl CoA oxidase activity was not affected by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. In the kidney, isozyme B of L-alpha-hydroxyacid oxidase activity was reduced by thyroidectomy and the diminished enzyme activity was restored by T4 administration in both 20- and 40-day-old rats. D-Amino acid oxidase and catalase activities in kidney, however, were not significantly modified by thyroidectomy or by T4 administration in either 20- or 40-day-old rats. The results suggest that thyroid hormone can modify the peroxisomal enzyme activity, which is prominent in immature animals.     

N-Acetylneuraminic acid biosynthesis in rat liver and kidney

Rat liver and kidney tissue slices incubated withN-acetyl [³H]mannosamine incorporated radioactivity into free and boundN-acetylneuraminic acid and CMP-N-acetylneuraminic acid (CMP-NeuAc). Liver and kidney also incorporated radioactivity from intravenously injected [³H]ManNAc intoN-acetylneuraminic acid and CMP-NeuAc. From the decrease in the specific radioactivity of CMP-NeuAc after a single injection ofN-acetyl[³H]mannosamine the half-life of CMP-NeuAc was determined. From this half-life and the pool size of CMP-NeuAc a synthesis rate of CMP-NeuAc was calculated, being 1.2 nmol/min/g wet weight of kidney. In previous experiments a value of 1.0 nmol/min/g wet weight was determined for liver [Ferwerdaet al. (1983) Biochem J 216: 87–92]. The synthesis rate of CMP-NeuAcin vivo was in the same range as the synthesis rate calculated from the turnover of boundN-acetylneuraminic acid, which was 2.7 and 0.4 nmol/min/g wet weight for liver and kidney respectively.The assay conditions for UDP-N-acetylglucosamine 2-epimerase andN-acetylmannosamine kinase were adapted to measure low activitiesin vitro. It appeared that the kinase activity detected in kidney can synthesizeN-acetylmannosamine6-phosphate at a rate sufficient for the observed production ofN-acetylneuraminic acidin vivo. Also a low, but measurable activity of UDP-N-acetylglucosamine 2-epimerase was detected in kidneyin vitro, suggesting that the biosynthetic pathway ofN-acetylneuraminic acid in kidney is the same as in liver. The synthesis rate ofN-acetylneuraminic acid in liver determinedin vivo is approximately 12 times slower than the maximal potential rate calculated from the activities of theN-acetylneuraminic acid (precursor-) forming enzymes as detectedin vitro. This indicates that in liverin vivo the enzymes are working far below their maximal capacity.     

Hormonal regulation of ornithine aminotransferase biosynthesis in rat liver and kidney.

The relative rates of ornithine aminotransferase (OAT) synthesis in vivo were studied by pulse-labeling rats with [4,5-³H]leucine, isolating the mitochondrial enzyme protein by immunoprecipitation with a monospecific antibody, dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels, and determining the radioactivity in OAT. After 4 days of treatment with triiodothyronine (T₃), both the enzyme activity level and the relative synthetic rate of OAT in rat kidney were elevated over twofold. The level of hepatic OAT activity was unaffected by this treatment. Thyroidectomy caused a 50% drop in the basal level of OAT activity and synthesis in kidney but not in liver. Although the basal levels of activity and synthesis of both renal and hepatic OAT were unaffected by adrenalectomy, the glucagon induction of the enzyme in liver was enhanced by about one-third and the T₃ induction in kidney was suppressed 50% by this operation. After 4 days of treatment with estrogen, both the enzyme activity level and the relative synthetic rate of OAT in male rat kidney were elevated nearly 10-fold. Hepatic OAT activity and synthesis were unaffected by this regimen. Thyroidectomy almost completely abolished the estrogen induction of OAT in kidney. OAT induction by estrogen could be restored by treating thyroidectomized rats with T₃. Simultaneous administration of T₃ plus estrogen to intact rats produced a multiple effect, resulting in a striking 20-fold induction of renal OAT. Although administration of either T₃ or estrogen causes an increase in the synthesis of immunoprecipitable OAT protein in rat kidney, each of these hormones may induce OAT by a different mechanism.     

Microsomal inducers of drug-metabolizing enzymes suppress cytidine nucleotide biosynthesis in rat liver.

The biosynthesis of cytidine nucleotides of liver rRNA was studied following single administration of seventeen different compounds. Only the substances recognized as inducers of the mixed-function oxidases of liver microsomes decrease the utilization of [2-¹⁴C]orotic acid for the synthesis of rRNA cytidine nucleotides.     

The biosynthesis of metallothionein in rat liver and kidney after administration of cadmium

The biosynthesis of the cadmium-binding protein, metallothionein, was studied in rat liver and kidney after injection of cadmium chloride. A simplified procedure for the isolation of metallothionein from liver and kidney tissues was devised. It was found that the concentration of a subcutaneously injected dose of 30 μmoles of ¹⁰⁹CdCl₂/kg in the liver reached the maximum within 36 h. Thereafter, a slow decrease in the concentration of the isotope was noted during the 3 week period. In the kidney, the isotope was taken up in two phases. During the first phase the uptake was faster and lasted for about 4 days. The second phase of ¹⁰⁹Cd accumulation showed a slower increase in the concentration of the isotope. In both liver and kidney tissues 75–80% of the ¹⁰⁹Cd was associated with metallothionein. Amino acid incorporation studies revealed that active biosynthesis of metallothionein took place in the kidney as well as in the liver of cadmium-exposed rats. The turnover of ³⁵S-labeled metallothionein was also investigated and the half-lives of the hepatic and the renal metallothionein were found to be 2.8 and 5 days, respectively.     

Effect of castration on purine biosynthesis in the rat liver and kidney

With the present study, the effects of testosterone deficiency on the biosynthesis of purine nucleotides and nucleic acids in rat liver and kidney has been investigated. 20% homogenates were prepared in 1 N HCl04, centrifuged and the nucleic acid were extracted from the precipitate with 0.7 N PCA (15 min at 90 degrees C): the acid-soluble purines were precipitated with 1 M AgNO3 from the supernatant of the centrifuged homogenate, then extracted with 1 M HC1. An increase of the specific activity of the liver (A, +49%; G, +47%) and of the kidney (A, +54%; G, +34%) has been observed (P less than 0.05), while no variation of the specific activity of nucleic acids was evident after castration.     

The nucleotide sequence of rat liver glycogen phosphorylase cDNA.

The nucleotide sequence of a cDNA coding for rat liver glycogen phosphorylase has been determined. The 2715 base pairs of the cDNA are sufficient to encode the total protein as determined by comparison with the liver type of glycogen phosphorylase of man. Human and rat liver glycogen phosphorylase showed 86% homology at the DNA level whereas the deduced amino acid sequence has 93.5% identity.     

The purine nucleotide cycle and ammoniagenesis in rat kidney tubules

The contribution of the purine nucleotide cycle to renal ammoniagenesis was examined in cortical tubule suspensions prepared from acidotic rats and incubated with [alpha-15N]glutamine, [15N]glutamate, or [15N]aspartate. Labeling of ammonia and adenine nucleotides was determined after enzymatic transformations designed to circumvent the technical problem that 15NH3 and H2O have the same nominal mass. Labeling of the adenine nucleotide was undetectable (less than 10%) even after 1 h of incubation. From the measured concentrations of adenine nucleotides and ammonia and the labeling of the ammonia, the flux through the purine nucleotide cycle was calculated to account for less than 1% of the deamination of alpha-amino groups from all three substrates. The glutamate dehydrogenase reaction is therefore the likely pathway for deamination. The rate of 15NH3 production from [alpha-15N]glutamine was two or three times greater than from added [15N]glutamate, indicating a preference for intracellularly generated glutamate. 15NH3 production from added [15N]aspartate was similar to and perhaps slightly greater than that from added [15N]glutamate.     

The nucleotide sequence of rat liver tRNAAsn

The major species of asparagine specific tRNA was isolated from rat liver, degraded to oligonucleotides, and shown to have the nucleotide sequence pG-U-C-U-C-U-G-U-m¹G-m²G-C-G-C- A-A-D-C-G-G-D-X-A-G-C-G-C-m²G-ψ-ψ-C-G-G-C-U-Q-U-U-t⁶A-A-C-C-G- A-A-A-G-m⁷G-D-U-G-G-U-G-G-Z-ψ-C-G-m¹A-G-C-C-C-A-C-C-C-A-G-G-G- A-C-G-C-C-A_OH. Although this tRNA contains several modified nucleotides in their expected positions, it is unique in having X, 3-(3-Amino-3-carboxy-n-propyl)uridine in loop I rather than in loop III; Q, 7-(4,5-cis-dihydroxyl-1-cyclopenten-3-yl-aminomethyl)-7-deazaguanosine in the wobble position of loop II; and Z, an unknown, and presently uncharacterized nucleoside, at position 23 from the 3′ terminus usually occupied by ribothymidine.