Letter: Mechanism of decarboxylation of 1,3-dimethylorotic acid. A possible role for orotate decarboxylase.
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Carboxy-Lyases↗
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Activation of orotidine 5'-monophosphate decarboxylase by phosphite dianion: the whole substrate is the sum of two parts.
We report that the binding of phosphite dianion to orotidine 5'-monophosphate decarboxylase (OMPDC) results in an 80 000-fold increase in kcat/Km for decarboxylation of the truncated substrate, 1-(beta-d-erythrofuranosyl)orotic acid (EO), which lacks a 5'-phosphodianion moiety. The intrinsic binding energy (IBE) of phosphite dianion in the transition state is 7.8 kcal/mol, which represents a very large fraction of the 11.8 kcal/mol IBE of the phosphodianion group of the natural substrate orotidine 5'-monophosphate (OMP). The data give kcat = 160 +/- 70 s-1 for turnover of EO in the active site of OMPDC containing phosphite dianion, which is significantly larger than kcat = 15 s-1 for turnover of OMP. Despite the weaker binding of the individual EO and HPO32- "parts" (KmKd = 0.014 M2) than of OMP (Km = 1.6 x 10-6 M), once bound, OMPDC provides a slightly greater stabilization of the transition state for reaction of the parts than of the whole substrate. Thus, the covalent connection between the reacting portion of the substrate and the nonreacting phosphodianion group is not necessary for efficient catalysis. This implies that a major role of the phosphodianion group of OMP is to provide binding interactions that are used to drive an enzyme conformational change, resulting in formation of an active site environment optimized for transition state stabilization.
Anions↗
Allopurinol challenge tests performed before and after living-related donor liver transplantation in citrullinaemia.
We performed allopurinol challenge tests to evaluate the metabolic state of a citrullinaemic patient who received a living-relative donor liver transplant. Before transplantation, large amounts of orotic acid and orotidine were excreted during the challenge test. Following transplantation, excretion of these compounds in response to allopurinol was normalised. The challenge test was a safe and useful method to evaluate the metabolic state of the patient.
Allopurinol↗
Regulation of xanthine dehydrogenase in chick liver. Further experiments on the effects of inosine, actinomycin D and other factors.
1. It has been confirmed that the xanthine-dehydrogenase activity of chick liver is enhanced by starvation and by administration of inosine; the effects of these treatments are not additive. 2. Inosine has no effect when given to chicks depleted of the enzyme by feeding a low-protein diet. 3. Actinomycin D prevents the effect of inosine, but itself enhances the activity of xanthine dehydrogenase. 4. The xanthine-dehydrogenase activity is unchanged after addition of orotic acid to the diet, and is stimulated by injection of inorganic iron.
Acids↗
The effect of tannic acid on the synthesis of protein and nucleic acid by rat liver.
1. As early as 1hr. after the intraperitoneal administration of tannic acid to rats, it could be demonstrated in the liver. At 3hr. the nuclear fraction contained the largest amount of tannic acid. 2. Nuclear RNA synthesis was inhibited in vivo 2hr. after the administration of tannic acid. Induction by cortisol of tryptophan pyrrolase was 90% inhibited at 24hr. 3. Incorporation of [1-(14)C]leucine into protein by liver slices from treated rats was decreased by 50% after 24hr. Its incorporation into postmitochondrial supernatant from treated animals was not inhibited. Incorporation into slices and postmitochondrial supernatants were inhibited in vitro by tannic acid. 4. The sequence of events: concentration of tannic acid in nuclei, inhibition of nuclear RNA synthesis, inhibition of protein synthesis and production of necrosis, is discussed.
Animals↗
RNA synthesis in regenerating mouse liver evaluated by incorporation of [methyl-14C]methionine and by determination of RNA polymerase activity.
The synthesis of RNA during mouse liver regeneration was studied by two different methods at 24 and 48 h after partial hepatectomy. Total chromatin-bound RNA polymerase activity showed an increase of 32% at 24 h after partial hepatectomy. At 48 h a slight increase in total activity was also observed in regenerating liver, but the difference was not significant. The increase in total RNA polymerase activity was due to a rise in RNA polymerase I plus III activity. This enzyme activity was increased at both 24 and 48 h. The increase was 57% at 24 h and 51% at 48 h. When [methyl-14C]methionine was used for labelling of methyl groups in rRNA, there was an increased specific radioactivity of this class of RNA at both 24 h and 48 h. The increases were 263 and 103% at 24 and 48 h respectively. Thus both methods revealed an increased synthesis of rRNA during mouse liver regeneration. The results are discussed in relation to previous results from this laboratory [Yngner, Carlberg, Lewan & Engelbrecht (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1069-1074; Yngner, Engelbrecht, Lewan & Annerfeldt (1979) Biochem. J. 178, 1-8; Yngner, Bengtsson, Carlberg, Engelbrecht & Wieslander (1980) Exp. Cell. Biol. 48, 393-403], which have shown that the incorporation of orotic acid or uridine into RNA is not increased in mouse liver regenerating after partial hepatectomy.
Animals↗
Pyrimidine metabolism and sugar nucleotide synthesis in rat liver.
With radioactive precursors, the labelling kinetics of the soluble pyrimidine nucleotides and of RNA were measured in rat liver to determine the contribution of the metabolic flows through synthesis de novo and the salvage pathway. To separate and quantify all pyrimidine nucleotides, an h.p.l.c. technique was developed using anion-exchange chromatography and reversed-phase chromatography. The concentrations of cytidine nucleotides were in the range of 30-45 nmol/g wet weight, and the concentrations of the uridine phosphates and of the UDP-sugars were approx. 6 and 20 times higher respectively. After a single injection of [14C]orotic acid and of [3H]cytidine, the specific radioactivities were determined as a function of time. The 14C/3H ratio was calculated and gave a good indication of the involvement of the different flows. It could be concluded that UTP derived from synthesis de novo and from the salvage pathway is not completely mixed before being utilized. The flow of the salvage pathway is relatively more directed to RNA synthesis in the nucleus and that of synthesis de novo to cytoplasmic processes. For CTP it could also be concluded that the flow of the salvage pathway was relatively more directed to RNA synthesis in the nucleus. Because of the nuclear localization of the enzyme CMP-NeuAc (N-acetylneuraminate) synthase, special attention was paid to CMP-NeuAc. However, a conclusion about a location about the synthesis of CMP-NeuAc could not unequivocally be drawn, because of the small differences in 14C/3H ratio and the different values for the CDP-lipids.
Animals↗
Glucagon administration in vivo stimulates hepatic RNA and protein breakdown in fed and fasted rats.
Liver RNA and protein breakdown rates were measured simultaneously in fed and in 24 h-fasted rats during a short-term cyclic perfusion, 1 h after an intraperitoneal injection of glucagon or of saline. RNA was labelled in vivo by an intraperitoneal injection of [6-14C]orotic acid, 60 h before the start of the perfusion. The accumulation of radioactive cytidine and valine in the perfusion medium for 15 min was used to determine RNA breakdown and proteolysis respectively. The portal glucagon/insulin ratio was significantly higher in the fasted glucagon-treated rats than in their fed counterparts. Although glucagon administration significantly increased RNA and protein degradation rates in the fasted and in the fed groups, the effect was greater after 24 h of starvation. The relationship between these biochemical changes and the alterations of the hepatocyte lysosomal system was investigated by determining the fractional cytoplasmic volume of lysosomal structures (autophagic vacuoles and dense bodies) by morphometry in the fasted glucagon-treated rats and in their controls. Hyperlucagonaemia significantly enhanced the relative volume of autophagic vacuoles without affecting that of dense bodies. The results showed that hyperglucagonaemia induced in vivo stimulated both liver RNA and protein breakdown and that this effect was modulated by the nutritional status of the rats.
Animals↗
In vivo cholesterol synthesis by the rat digestive tract. III. Evaluation of modulating factors.
Sterol radioactivity was measured in the gastrointestinal tract of rats fed a semi-purified basal diet (B), either enriched with 0.5% cholesterol (CH) or containing 2% orotic acid (O). These measurements were taken after a fast of 48 h (CHF) or after fasting and 4-aminopyrazolopyrimidine (APP) treatment (CHFA); the five groups were killed 70 min after a subcutaneous injection of [1-14C]-acetate. Since these results agree with current published data, it is suggested that, although this method is not quantitative, it can give accurate estimates of the qualitative variations of cholesterogenesis in one organ. Adding cholesterol to the diet had no effect on sterogenesis in the stomach and caecum-colon. Fasting for 48 h did not affect cholesterogenesis in the caecum-colon, but stomacal sterogenesis was reduced (50%). APP treatment, which did not affect cholesterogenesis in the stomach, strongly stimulated (4-fold) cholesterogenesis in the caecum-colon. A slight decrease (30%) in intestinal cholesterogenesis was observed after a cholesterol-rich diet. This decrease occurred mainly in villus enterocytes. Fasting reduced cholesterol synthesis 2 to 4-fold mainly in the proximal intestine, while APP treatment stimulated it until a level higher than in nourished control rats. The level of cholesterogenesis was similar in all the enterocytes collected from duodenum to ileum in APP-treated rats. Since there was high mucosa cell loss (about 50%) during the 48-hour APP treatment, total intestinal cholesterogenesis in the CHFA rats was not higher than in the CH animals. Under the present physiological conditions, the feedback inhibition of intestinal cholesterogenesis by luminal bile acids was not clear, while that by luminal cholesterol or by LDL-cholesterol penetrating by specific receptors was modest over a wide range of physiological conditions.
Animals↗
Characterization of the rodent genes for arylacetamide deacetylase, a putative microsomal lipase, and evidence for transcriptional regulation.
In the current study, we have determined the cDNA and the genomic sequences of the arylacetamide deacetylase (AADA) gene in mice and rats. The AADA genes in the rat and mouse consist of five exons and have 2.4 kilobases of homologous promoter sequence upstream of the initiating ATG codon. AADA mRNA is expressed in hepatocytes, intestinal mucosal cells (probably enterocytes), the pancreas and also the adrenal gland. In mice, there is a diurnal rhythm in hepatic AADA mRNA concentration, with a maximum 10 h into the light (post-absorptive) phase. This diurnal regulation is attenuated in peroxisome proliferator-activated receptor alpha knockout mice. Intestinal but not hepatic AADA mRNA was increased following oral administration of the fibrate, Wy-14,643. The homology of AADA with hormone-sensitive lipase and the tissue distribution of AADA are consistent with the view that AADA plays a role in promoting the mobilization of lipids from intracellular stores and in the liver for assembling VLDL. This hypothesis is supported by parallel changes in AADA gene expression in animals with insulin-deficient diabetes and following treatment with orotic acid.
Acyl-CoA Oxidase↗
The role of cortisone acetate in liver RNA synthesis of gamma-irradiated rats.
The possible role of induced endogenous glucocorticoid levels stimulated by ionizing radiation and their subsequent effect on rat-liver RNA was investigated in adrenalectomized rats using an intraperitoneal injection of cortisone acetate immediately prior to exposure to gamma-radiation (1800 rad). Adrenalectomized rats were divided into four groups: (1) control rats, (2) irradiated rats, (3) nonirradiated rats treated with cortisone acetate (10 mg/rat), and (4) irradiated rats which received cortisone acetate immediately before irradiation. Newly synthesized RNA was measured by an intraperitoneal injection of 8 muCi (6-14C) orotic acid either immediately after irradiation or 40 min prior to killing. All the rats were killed 6 hours post-irradiation. The results of these investigations indicate that (1) newly synthesized (40 min labelling) RNA increased in irradiated adrenal-intact rats, but not in adrenalectomized animals; (2) cortisone acetate by itself stimulated RNA synthesis whether the radioactive precursor was administered 6 hours of 40 min prior to killing; and (3) irradiation of adrenalectomized rats pre-treated with cortisone acetate induced more RNA synthesis than did cortisone alone. The results of this study support the suggestion that ionizing radiation may induce rat-liver RNA synthesis, at least in part, via stimulated adrenal secretion of glucocorticoids.
Adrenal Glands↗
Effects of ionizing radiation and partial hepatectomy on messenger RNA synthesis.
Newly synthesized messenger RNA, as measured by a 40 min uptake of the radioactive precursor (6-14C) orotic acid, was studied in the regenerating livers of non-irradiated and gamma-irradiated (1800 rad) adrenal-intact and adrenalectomized rats 24 aand 48 hours after partial hepatectomy. Two groups of rats, one with and one without adrenal glands, were each divided into four subgroups: (1) control rats, (2) irradiated rats, (3) partially hepatectomized rats and (4) irradiated, partially hepatectomized rats. The radioactive profile of polyribosome formation and distribution was determined by sucrose density gradient centrifugation (10--40 per cent). The result of this study indicates that ionizing radiation decreases the synthesis of newly formed messenger RNA in re generating livers of adrenal-intact rats. However, adrenalectomy largely abolished that inhibition. These data suggest that the decrease in messenger RNA synthesis may be explained by the disturbance of adrenal hormones induced by partial hepatectomy and ionizing radiation.
Adrenal Glands↗
E.s.r. and spin-trapping studies of dihydropyrimidines. gamma-Radiolysis in the polycrystalline state and U.V. photolysis in aqueous solution.
gamma-Radiolysis in the polycrystalline state and U.V. photolysis in aqueous solution at 220 nm of several dihydropyrimidines and their derivatives have been investigated by spin-trapping and electron spin resonance. 2-Methyl-2-nitrosopropane was used as the spin-trap. The spin-adducts of the 6-yl radicals obtained fall into two categories. Those from dihydro-1-methyluracil, dihydro-6-methyluracil, dihydro-1-ethyluracil and dihydro-1-methylcytosine exhibit a beta-nitrogen hyperfine coupling constant (alpha beta N) equal to or less than 2.0 G while the ones fom dihydro-orotic acid, dihydrouracil and dihydrothymine showed much larger alpha beta N values (greater than 3.3 G). Dihydrouridine gives radicals characteristic of both the dihydropyrimidine ring and the sugar moiety. The same radicals were obtained by gamma-radiolysis or U.V. photolysis. For all the 5-yl radicals obtained by U.V. photolysis, a direct photoexcitation mechanism is proposed.
Crystallization↗
Membrane-bound ribosomes in kidney: methods of estimation and effect of compensatory renal growth.
Membrane-bound ribosomes are thought to secrete protein for export and free ribosomes to secrete protein for intracellular use. The proportion of the total ribosomes that is bound to membranes in normal mouse kidneys has been estimated by three different methods, and the results have been compared with those obtained by a fourth method used by us previously. The most valid estimates appear to be those obtained (a) by comparison of radioactivity in peaks representing free and membrane-bound ribosomes on linear sucrose gradients after labeling for 24 hr with (14)C-orotic acid, and (b) by measurements of optical density in free and bound ribosomes that had been separated by centrifugation on discontinuous gradients of 0.5 M/2.0 M sucrose. Analyses by these methods show that about 20-25% of the ribosomes in a postnuclear supernatant prepared from mouse kidneys, but only 10-15% of the ribosomes in a post-mitochondrial supernatant, are membrane-bound. About 75% of the bound ribosomes sediment as polysomes of many different sizes. The proportion of membrane-bound ribosomes and their aggregation into polysomes were unchanged in kidneys undergoing compensatory hypertrophy after removal of the opposite kidney. These experiments show that, unlike liver, kidney has a predominance of free ribosomes compared to bound ribosomes; those ribosomes that are membrane-bound do not become free during compensatory renal growth.
Animals↗
Selective release of content from microsomal vesicles without membrane disassembly. I. Permeability changes induced by low detergent concentrations.
Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025-0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.
Animals↗
An early effect of diphtheria toxin on the metabolism of mammalian cells growing in culture.
At concentrations of 0.1 Lf (0.25 microg.)/ml., or greater, diphtheria toxin produces an immediate decrease (40 +/- 5 per cent) in the steady-state level of incorporation of inorganic phosphate into ATP by cultures of normal human kidney cells growing at 37 degrees C. in the presence of glucose. The effect is readily reversible by specific antitoxin for a period of 30 minutes or more after adding toxin. Protein synthesis in these cells continues at a normal rate for 60 minutes after adding toxin and the toxin-treated cells are able to take up and concentrate potassium ions normally for at least 1 hour. Even high concentrations of toxin (5 Lf/ml. or more) fail to effect either protein synthesis or ATP formation by cultures of spleen cells from the mouse, an animal that is relatively resistant to the lethal action of the toxin. Of various inhibitors studied, antimycin A most closely resembles toxin in its action, both on protein synthesis and on ATP formation. Mouse cells are considerably more resistant to antimycin A than are human kidney cells. Human kidney cells treated with saturating doses of toxin continue to form RNA at a normal rate for about 1 hour, after which the rates of both P(i) (32) incorporation and of 6-C(14)-orotic acid incorporation into RNA sharply decline and continue at about 40 to 50 per cent their initial rate. At 37 degrees C., S(35)-methionine incorporation into cell protein ceases altogether 60 to 75 minutes after addition of a saturating dose of toxin. The effect of saturating doses of toxin on S(35)-methionine incorporation into human kidney cell protein at different temperatures, has been studied. It is concluded from the present studies that diphtheria toxin exerts a primary, reversible effect at the surface of susceptible cells where it inhibits cytochrome-linked phosphorylation concerned with transport of inorganic phosphate across the cell membrane.
Animals↗
Evaluation of the transfer and expression in mice of an enzyme-encoding gene using a human adenovirus vector.
Mutant mice of the Spf-ash strain have an inherited defect in ornithine transcarbamylase (OTC) protein synthesis, and were used to ascertain the potential of recombinant adenoviruses for introducing and expressing the normal gene lacking in these mice. These OTC mutant mice are characterized by a reduction in the amount of OTC activity, resulting in hyperammonemia, pronounced orotic aciduria, growth retardation, and sparse fur until weaning. A recombinant adenovirus that harbors the rat OTC cDNA under the control of the viral major late promoter (MLP) was constructed and injected into such newborn mice. The effect of the virus was analyzed by monitoring the hepatic OTC enzyme during several months after the injection. An increase in OTC activity was detected and was accompanied by a diminution of orotic acid in the urine. The observation of MLP-OTC mRNA transcripts over 1 year following the injection attests to the relatively long-term presence of the transferred gene. In those mice showing the greatest OTC activity, a normalization of the fur was also observed. The experiments reported here document the feasibility of using adenovirus for the direct delivery in vivo of a gene to restore an impaired metabolism.
Adenoviruses, Human↗
Picolinic acid in milk, pancreatic juice, and intestine: inadequate for role in zinc absorption.
Picolinic acid (PA) was measured by high pressure liquid chromatography in human milk and other fluids and tissues. Skimmed human milk, intestinal homogenates from human infants and rats, and human and rat pancreatic juice were ultrafiltered and analyzed by high pressure liquid chromatography using an anion-exchange column. Identity of sample components was verified by comparing retention times with those of pure nicotinic acid and PA. The detection limit for PA was 2.5 microM. Human milk contained less than 3.7 microM PA. PA was undetectable in human infant or rat intestine or in human or rat pancreatic juice. The extremely low concentration of PA in milk and its apparent absence in pancreatic juice and intestine provide additional evidence that PA is not the low molecular weight zinc binding ligand of human milk and that it does not have an important physiological role in intestinal zinc absorption.
Animals↗