Alois Cihak

The effect of pyrimidine analogues and tryptophan on enzyme synthesis and degradation in rat liver

Summary 5-Fluoroorotic acid, when administered from the zero time point, results in a marked inhibition of the dietary induction of four amino acid-degrading enzymes in rat liver (serine dehydratase, ornithine transaminase, histidase, and tryptophan oxygenase). When tyrosine aminotransferase is induced by the same technique, however, the administration of 5-fluoroorotic acid results in a marked increase in the induced levels of the enzyme. 5-fluoroorotic acid and another pyrimidine analogue, 5-azacytidine, were also shown to stimulate hormonal induction of tyrosine aminotransferase. 5-Fluoroorotic acid was found to be the most potent of several orotic acid analogues studied in the inhibition of the dietary induction of serine dehydratase, and 5-azacytidine was much more effective than 5-fluoroorotic acid on a mg basis. Both analogues also inhibit the hormonal induction of serine dehydratase, and appear to be acting in a manner similar to actinomycin D in that inhibition of enzyme induction occurs only when the administration of the inhibitors is not delayed significantly after the inducers are given. 5-Fluoroortic acid does not inhibit the further induction of tyrosine aminotransferase by cortisone after initial induction by casein hydrolysate in intact animals, but rather prevents the decay of the induced level of the enzyme seen in animals given cortisone alone. In adrenalectomized animals, 5-fluoroorotic acid delays the decay of tyrosine aminotransferase levels induced by casein hydrolysate alone, and actually stimulates the “super-induction” by cortisone. Using immunochemical techniques, it was shown that the enhancement of the hormonal induction of tyrosine aminotransferase by 5-fluoroorotic acid and 5-azacytidine was not related to an increased rate of synthesis, but rather to stabilization of the enzyme. Tryptophan, another efficient inducer of tyrosine aminotransferase, also appears to exert its effect by stabilizing enzyme turnover. The incorporation of 5-fluoroorotic-2-C 14 acid into hepatic RNA and ribonucleoprotein was studied in an effort to gain some insight as to the mechanisms by which this compound influences enzyme induction. 5-fluoroorotate-2-C 14 -labeled cytoplasmic RNA, recently shown to be nonribosomal by chromatographic and sedimentation criteria ( 17 ), was further characterized by studying the polysome-associated ribonucleoprotein in which it is found. The radioactive analogue appears to label selectively a population of ribonucleoprotein which is heterodisperse, nonribosomal, with respect to both sedimentation coefficient and density. The incorporation of 5-fluoroorotic acid into this population of ribonucleoprotein is relatively insensitive to actinomycin D. Possible mechanisms linking base analogues and tryptophan to enzyme turnover are discussed.

Studies on the mechanism of the stimulation of tyrosine aminotransferase activity in vivo by pyrimidine analogs: the role of enzyme synthesis and degradation.

Abstract The pyrimidine analogs, 5-fluoroorotate and 5-azacytidine, have been shown to stimulate the basal level as well as the cortisone, tryptophan, and casein hydrolysate-induced levels of the rat liver enzyme, tyrosine aminotransferase. This stimulation was most marked in the case of dietary and hormonal induction when the analog was given 4–6 hr prior to the administration of the inducer. When tryptophan induced tyrosine aminotransferase, maximal stimulation by the analog occurred if it were given 2 hr prior to the administration of the amino acid. The optimal stimulatory dose of 5-azacytidine was 5 mg/kg body weight whereas 5-fluoroorotate gave its highest stimulation at a dose of 60 mg/kg. Of several orotic acid analogs tested, only the chloro-analog had an effect similar to the fluoro-congener. Utilizing quantitative immunochemical precipitation and pulse labeling in vivo, it was demonstrated that the administration of 5-fluoroorotate or 5-azacytidine at doses of 60 and 5 mg per kg, respectively, while causing a stimulation in the basal level of tyrosine aminotransferase, did not result in any change in the rate of enzyme synthesis. Furthermore, after cortisone induction of the enzyme, the delayed administration of these analogs caused either a further stimulation in the level of the enzyme or the maintenance of a high level while the enzyme activity decayed in animals not given the analogs. The rates of synthesis either showed no change or a decrease while the amount of enzyme was increasing. Prelabeling of the enzyme in vivo after induction with cortisone and followed by the administration of 5-fluoroorotate resulted in a marked decrease in the t 1 2 of the decay rate of the enzyme measured either by loss of radioactivity or by loss of enzyme activity. These studies suggest that these analogs act in some manner to prevent enzyme turnover by an inhibition of enzyme degradation.

l-Tryptophan inhibition of tyrosine aminotransferase degradation in rat liver in vivo☆

Abstract The administration of l -tryptophan to both intact and adrenalectomized animals results in a marked increase in the activity of tyrosine aminotransferase. Maximal increases in enzyme activity are stimulated by doses of l -tryptophan much lower than those required for maximal stimulation of tryptophan oxygenase activity in vivo . When l -tryptophan was administered to animals that had been given cortisone 5 hr earlier, a further sustained increase in enzyme activity was demonstrated. 5-Hydroxy- dl -tryptophan and indole administration in amounts equimolar to l -tryptophan also result in similar increases in activity whereas α-methyl- dl -tryptophan produces little or no increase. Utilizing pulse-labeling in vivo with quantitative immunochemical precipitation of tyrosine aminotransferase by specific antisera, it was demonstrated that the administration of tryptophan caused an increase in enzyme amount with no concomitant increase in the rate of enzyme synthesis. In animals given cortisone, subsequent injections of tryptophan caused the amount of enzyme to continue to increase while both the amount of enzyme in control animals, as well as the rates of synthesis in both tryptophan-treated and control animals, decreased in a parallel fashion. Prelabeling of tyrosine aminotransferase in vivo after the enzyme had been induced with cortisone demonstrated that the subsequent administration of tryptophan caused a marked inhibition in the decay of the radioactive enzyme, as well as in enzyme activity. These data support the proposal that the amino acid, tryptophan, has a special role both in the maintenance of hepatic protein synthesis and in the regulation of specific enzyme degradation in rat liver.

Effect of 5-azacytidine on dietary and hormone induction of serine dehydratase and tyrosine aminotransferase in rat liver

Abstract Dietary and hormone induction of serine dehydratase and tyrosine aminotransferase in the liver of rats treated with 5-azacytidine has been studied by a specific immunoprecipitation technique. While the synthesis of serine dehydratase induced by cortisone or dietary tryptophan is depressed by 5-azacytidine administered simultaneously or after the inducer, the increase of tyrosine aminotransferase is not affected by the analogue administered after the inducer. 5-Azacytidine does not block the increase of tyrosine aminotransferase once it has started; moreover, the drug alone causes a similar enhancement of the enzyme activity. However, induction of tyrosine aminotransferase is partially depressed by the simultaneous administration of the drug with the inducer. The significance of the findings in view of the known inhibitory effect of 5-azacytidine is discussed.

Incorporation of label from 5-fluoroorotate into non-ribosomal cytoplasmic RNA in rat liver

Studies from several laboratories, notably that of Heidelberger [ 1,2] , and others [3] have demonstrated that upon administration of radioactively labeled 5fluoroorotic acid in vivo, the label occurs in free pyrimidine nucleotides in liver and is subsequently incorporated into pyrimidine nucleotide residues of RNA. However, few studies have been carried out in mammalian tissues on the actual types of RNA within the hepatic cell that are labeled by fluoropyrimidines. The studies described in this brief communication demonstrate that administration of 2-14C-5-fluoroorotic acid leads to the labeling of RNA which has none of the characteristics of ribosomal RNA and a number of the characteristics of cytoplasmic “messenger” RNA as described for mammalian systems [4-61.

Studies on the induction and repression of enzymes in rat liver: Characteristics of the l-tryptophan and cortisone-mediated induction of serine dehydratase in the livers of intact and adrenalectomized rats☆☆☆

Abstract Liver serine dehydratase differs in its response to the administration of cortisone and l -tryptophan in intact and adrenalectomized rats. While the enzyme synthesis de novo is increased by the administration of tryptophan or cortisone in intact rats, cortisone but not tryptophan stimulates synthesis of the enzyme in adrenalectomized animals. The magnitude of the enzymes increase after tryptophan administration depends on the protein content of the diet and the length of starvation of the animals prior to the induction. While the combination of cortisone with tryptophan does not result in a further enhancement of hepatic serine dehydratase in rats with intact adrenal glands, glucagonmediated induction of the enzyme in these animals can be further increased by giving the amino acid. No such effect was found in adrenalectomized animals. Both the increase of serine dehydratase activity caused by tryptophan in intact, and by cortisone in adrenalectomized rats is associated with the appearance of form I of serine dehydratase as well as an increase in the total activity of the enzyme.

Enhancement of rat liver uridine kinase activity by various metabolic inhibitors

Abstract Of 25 compounds tested, eight—namely, 5-azacytidine, gougerotin, cycloheximide, pactamyein, streptovitaein A, adriamyein, daunoruhicin and thioacetamide resulted in the enhancement of liver uridine kinase actiuty in vivo . Puromycin and actinomycin D produced a slight decrease in enzyme activity. With both 5-azaeytidine and cyeloheximide, the enhancement observed was independent of adrenal secretion. Some of the compounds which enhanced hepatic uridine kinase activity 15-azacytidine, cycloheximide and related glutarimide antibioties concomitantly suppressed the activity of the enzyme in the thymus, while daunoruhicin adriamycin and thioacetamide were much less effective in this respect. No relation has been found between the effect of the tested compounds on the incorporation of orotic acid into RNA and the enhanced activity of hepatic uridine kinase.

Depression of DNA Synthesis in Mouse Spleen After Treatment With 5-Aza-2′-deoxycytidine

5-Aza-2-deoxycytidine is a highly effective cytostatic agent that preferentially affects the lymphatic system. Pretreatment of noninbred H mice with the drug markedly depressed the level of thymidine (dThd) incorporation into DNA in the spleen and also lowered the dThd and thymidylate kinase activities. Maximum effects were observed following administration of the analog in a single dose 24 hours before the mice were killed. Whereas cytidine and dThd did not reverse the inhibitory effect of 5-aza-2-deoxycytidine, excessive doses of deoxycytidine partially reversed this inhibition. Similar to the depression of dThd incorporation, a depression in the incorporation of deoxycytidine and cytidine into spleen DNA was found after 24-hour pretreatment with 5-aza-2-deoxycytidine. However, 7 days following 5-aza-2-deoxycytidine treatment, the incorporation of dThd into DNA in the spleens of mice was significantly increased. [3H]5-aza-2-deoxycytidine was rapidly incorporated into spleen DNA, whereas deoxycytidine interfered with the incorporation of [3H]5-aza-2-deoxycytidine.