Adenylate Deaminase Activity Research Articles

Antacids’ side effect hyperuricaemia could be alleviated by long-term aerobic exercise via accelerating ATP turnover rate

Hyperuricemia is the term for an abnormally high serum uric acid level. Many factors contribute to hyperuricemia, however no definite correlation between proton pump inhibitors (PPIs) and hyperuricemia has been reported before. Physical exercise also decreases serum uric acid levels. However, the detailed biochemical-regulatory mechanisms remain unknown. Here we found that adenylate deaminase activities are much higher in hyperuricemia patients than in the healthy people. Therefore, the patients have higher levels of adenosine metabolites hypoxanthine and uric acid. Acid-inhibitory drugs (antacids) significantly increased serum uric acid level and may lead to gout in the hyperuricemia patient. Long-term aerobic exercise significantly increased serum phosphorus and decreased serum ATP and its metabolites, and therefore decreased serum uric acid. Antacids slow down the ATP turnover rate and result in serum uric acid elevation subsequently. While the long-term aerobic exercise decreases serum uric acid levels by accelerating ATP turnover rate. The results imply that long-term aerobic exercise may be a useful strategy to prevent and treat hyperuricaemia.

Effects of alcohol–kola nut interactions on brain glucose oxidase, glutamine synthase, and adenylate deaminase activities in Wistar rats

The effects of alcohol–kola nut interactions on activites of whole brain glucose oxidase, glutamine synthetase, and adenylate deaminase were examined in Wistar rats. Thirty Wistar albino rats were divided into six groups. Control group (1) received a placebo (4 mL of distilled water). Groups 2–6 were treated for a 21-day period with either 10% (v/v) alcohol, kola nut, caffeine, alcohol + kola nut, or alcohol + caffeine at 50 mg per kg in a total volume of 4 mL vehicle via gastric intubation, respectively. One day after the final treatment, the whole brain was harvested and processed to examine for several biochemical parameters, including activities of glucose oxidase, adenylate deaminase, and glutamine synthetase. Results showed that alcohol–kola nut interaction decreased the activities of glucose oxidase, adenylate deaminase, and glutamine synthetase in whole brain, suggesting inhibition of glutamine synthesis, decreased energy (ATP) production, ionic transport and decreased neuronal processes. Data indicates that alcohol–kola nut or alcohol–caffeine interactions modify neuronal processes in rat brain; however, the clinical importance of this is not known.

ChemInform Abstract: 2′,3′‐Isopropylidene Group, a Molecular Scaffold to Study the Activity of Adenosine and Adenylate Deaminase on Adenosine Analogues Modified in the Ribose Moiety

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Activity of Adenosine Deaminase and Adenylate Deaminase on Adenosine and 2', 3'-Isopropylidene Adenosine: Role of the Protecting Group at Different pH Values

The deamination rate of 2',3'-isopropylidene adenosine catalyzed by adenosine deaminase (ADA) from calf intestine and adenylate deaminase (AMPDA) from Aspergillus species has been evaluated and compared with that of the enzymatic reactions of adenosine, to elucidate the influence of the protecting group on ezyme activity.

2′,3′-Isopropylidene Group, A Molecular Scaffold to Study the Activity of Adenosine and Adenylate Deaminase on Adenosine Analogues Modified in the Ribose Moiety

2 ′,3 ′-Isopropylidene group can be used as a molecular scaffold for the introduction of modifications at 5 ′ and 1 ′ positions of adenosine and these modified nucleosides are used to evaluate the biocatalytic activity of adenosine and adenylate deaminase.

Vitamin A Deficiency in Mice Enhances the Colonic Level of Purine Enzyme Activity

Background: Vitamin A is an important nutritional factor that regulates normal growth and functions of epithelial cells of the gastrointestinal tract. Objective: The objective of this study was to examine the role of vitamin A on the histological and biochemical changes in the colon of mice. Methods: To address this issue, vitamin A deficiency (VAD) was developed in mice by placing them on a VAD diet from weaning up to 120–170 days. Infiltration of inflammatory cells in the colon was determined histologically. Activities of adenosine deaminase, adenylate deaminase, purine nucleoside phosphorylase and myeloperoxidase were determined. Results: VAD in mice induced a significant increase in the number of mast cells per 100 crypts. There was also an abundance of other connective tissue cells such as plasma cells, lymphocytes and neutrophils around the crypts in the lamina propria. The colonic activity of adenosine deaminase and adenylate deaminase was increased due to VAD, whereas purine nucleoside phosphorylase activity remained unchanged. Immunohistochemical analysis showed an increased expression of adenosine deaminase in VAD mice colon. The increase in myeloperoxidase activity was not statistically significant. Conclusions: VAD causes upregulation of purine enzyme, which together with an increased number of inflammatory cells might exacerbate colonic injuries in VAD condition.

Age-related changes of AMP breakdown in chicken heart

The activity of adenylate deaminase, adenylate phosphatase and adenosine deaminase, as well as the endogenous content of adenine nucleotides, was examined in the heart of ageing chickens. In new-born (1-day-old) and young (20-day-old) chickens, AMP degradation in the heart seems to proceed preferentially through deamination, while in adult (1-year-old) through dephosphorylation. Compared with the adult heart, a 2-year-old one exhibits a decline of AMP catabolism. The total adenine nucleotide content and the concentration of ATP are higher in adult and aged chicken hearts, than in new-born and young ones. Adaptive mechanisms might occur in the heart of ageing chickens to ensure an adequate availability of adenine nucleotides.

Occurrence of the purine nucleotide cycle in rat pancreatic islets.

The presence of the purine nucleotide cycle is investigated in rat pancreatic islets. Adenylosuccinase, adenylate deaminase, and adenylosuccinate synthetase activities are characterized in islet homogenates. In the assay of the latter enzyme, evidence is obtained for operation of the full cycle in islet extracts. The activities of the three enzymes are not vastly different in islet and brain. These findings are discussed in the light of the role currently ascribed to the purine nucleotide cycle in producing ammonia from amino acids, in adjusting the concentration of Krebs cycle intermediates, in regulating the relative concentrations of ATP, ADP, and AMP, and in controlling the activity of phosphofructokinase.

Expression of different isoenzymes of adenylate deaminase in cultured human muscle cells. Relation to myoadenylate deaminase deficiency

Adenylate deaminase activity was determined in cultured muscle cells of different maturation grades and muscle biopsies from normal subjects and four patients with a primary myoadenylate deaminase (MAD) deficiency. Adenylate deaminase activity was much lower in cultured human muscle cells than in normal muscle. .The activity increased with maturation. The ratio of activities measured at 5 and 2 mM AMP decreased in the order: immature muscle cells > more mature muscle cells > muscel. Adenylate deaminase activity was detectable in muscle cell cultures of MAD-deficient patients. However, both at 2 and 5 mM AMP this activity was significantly lower than in cultured cells with the same high maturation grade obtained from control sujects, whereas the ratio between the activities at 5 and 2 mM AMP was higher. The observations indicate that transition from a fetal to an adult muscle isoenzyme deaminase takes place in human cultured muscle cells during maturation. In cultures obtained from MAD-deficient patients this transition does not occur and only the fetal isoenzyme is present.

Metabolic Adaptations to Nitrogen Excess in Late Gestation in Rat

Pregnant rats of 19th and 21st days were given an acute nitrogen overload produced by an infusion of either 0.2 M ammonium acetate or 0.2 M glutamine. Metabolic adaptations to nitrogen excess were studied measuring--in fetomaternal unit--non-protein nitrogen content and the activities of enzymes related with ammonia metabolism. Maternal and fetal plasma urea levels were increased by ammonium acetate treatment. Glutamine overload increased more the amino acid content in the mothers than in conceptus. As response to ammonium acetate treatment, glutamate dehydrogenase activity in liver was more sensitive in pregnant than in nonpregnant rats, suggesting more nitrogen incorporation into amino acids in pregnancy. Regarding glutamine synthetase activity, both treatments had an opposite effect except in kidney. The adenylate deaminase activity of pregnant rats was inhibited similarly to nonpregnant rats by nitrogen overloads, but stronger after glutamine infusion. Placenta and fetal metabolism were adjusted, as the dams, to lack of ammonia production by nitrogen overloads and to glutamine synthesis by ammonium acetate infusion.

Phorbol esters and cyclic AMP activate AMP deaminase in adult rat cardiac myocytes

Using rapid deenergization as a probe for adenylate deaminase activity in intact adult rat cardiac myocytes, we have previously established that IMP formation is enhanced by α-adrenergic agonists. In the present study, the effect of adrenergic agents on adenylate deaminase was further characterized. Phenylephrine (PE) 3 increased IMP production in a dose-dependent fashion with an EC 50 of 8 × 10 −7 m. The response to PE was reversed within 10 min by the α 1-antagonist, prazosin. Likewise, adenylate deaminase was also activated in ventricular myocytes challenged with phorbol 12-myristate 13-acetate (PMA, EC 50 = 5 n m); cardiac cells presented with 100 n m PMA increased IMP production from 4.4 ± 0.5 (control) to 15.7 ± 0.9 nmol/mg protein when subsequently deenergized. The effects of PMA and PE were attenuated 85 ± 5% and 96 ± 4%, respectively, by pretreatment of cells with 150 n m staurosporine, an inhibitor of protein kinase C. Furthermore, incubation of cardiac cells with 1 μ m PMA for 24 h blunted the response to both PMA and phenylephrine 85–90%. Elevating cyclic AMP (cAMP) content to >15 pmol/mg by treatment with forskolin or isoproterenol plus isobutylmethylxanthine also resulted in enhanced adenylate deaminase activity, but this stimulatory effect was not abolished by 24 h incubation with 5 μ m PMA. Forskolin and PMA-induced increases in IMP production appeared to be additive. However, 0.5 μ m isoproterenol inhibited the cellular response to phenylephrine by about 30% but did not affect PMA-stimulated adenylate deaminase activity. We conclude that both cAMP and protein kinase C stimulate adenylate deaminase, perhaps through selective activation of different isoforms. However, cAMP also exerts partial inhibition on α-adrenoreceptor-mediated increases in IMP production.

Purine and pyrimidine metabolism in human muscle and cultured muscle cells

Using radiochemical methods, we determined the activities of various enzymes of purine and pyrimidine metabolism in homogenates of human skeletal muscle and of cultured human muscle cells. Results show a large discrepancy between the enzyme activities in muscle and cultured cells. With regard to purine metabolism, adenylate (AMP) deaminase activity was only 1–3% in cultured cells compared to that in muscle, whereas the activity of adenosine deaminase, purine-nucleoside phosphorylase, adenosine kinase, adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase was 7–15-fold higher in the cultured cells. The enzymes of pyrimidine metabolism, orotate phosphoribosyltransferase, orotidine 5′-monophosphate decarboxylase and uridine kinase showed activity of 100–200-fold higher in cultured cells than in adult muscle. The differences in enzyme activity are probably related to the low differentiation stage and the absence of contractile activity in the cultured muscle cells. Care must be taken when using these cells as a model for studying purine and pyrimidine metabolism of adult myofibers.

Glutamine and ammonium handling by anaesthetized rats

The infusion of ether anesthaetized rats with 0.2 M (1 mmols in total) ammonium acetate or glutamine were compared with the infusion of 0.2 M NaCl. The levels of circulating glucose, amino acids, lactate, urea and ammonium were measured as well as liver glycogen and tissue amino acids and the liver and muscle activities of carbamoyl phosphate synthetases I and II, glutamate dehydrogenase, glutamine synthetase and adenylate deaminase. Neither treatment altered the glucose and glycogen homeostasis. The infusion of ammonium did not result in increases in circulating ammonium, but resulted in increased circulating urea after a short delay; the infusion of glutamine resulted also in urea production but much later on. Glutamine infusion also resulted in increased tissue free amino-acid levels. There was little alteration in enzyme activities, except for decreased glutamine synthetase and adenylate deaminase activity in muscle of glutamine-infused rats and higher tissue carbamoyl phosphate synthetase II. The results agree with a fast removal of infused ammonium, and maintenance of glutamine, with their channeling towards urea production at a rate comparable with that of infusion, that did not alter significantly the homeostasis of the experimental animals.

Effect of cold-temperature exposure and acclimation on amino acid pool changes and enzyme activities of rat brown adipose tissue

The amino acid pool composition and its concentration ratios with respect to blood and plasma, as well as the activities of alanine, aspartate and branched chain amino acid transaminases, glutamina synthetase, adenylate deaminase and glutamate dehydrogenase have been studied in the interscapular brown adipose tissue of control, 12-h cold-exposed and 15-day cold-acclimated rats. Cold temperature affected the amino acid metabolism and pool composition more intensely after 15 days than after 12-h cold-exposure, even though the patterns of change were very similar in both groups. Cold temperatures induced a decrease in glutamine and an increase in glutamate concentration in the tissue. This probably increased the metabolism of branched chain amino acids and caused a decrease in adenylate deaminase activity. It also seemed to increase alanine utilization. We concluded that amino acid metabolism in brown adipose tissue is enhanced by cold temperature acclimation.

Superiority of HPLC to Assay for Enzymes Regulating Adenine Nucleotide Pool Intermediates Metabolism: 5'-Nucleotidase, Adenylate Deaminase, Adenosine Deaminase, and Adenylosuccinate Lyase-A Simple and Rapid Determination of Adenosine

Abstract A new application of HPLC analysis to assay for all the enzymes involved in regulation of adenine nucleotide pool metabolism is described. In vitro, enzymatic reactions were carried out in buffered reaction media containing appropriate concentrations of metal ions, specific substrate, inhibitors and enzymes. Following an incubation period, the enzymatic reactions were terminated and extracted with cold trichloroacetic acid. The soluble acid extracts were neutralized and injected in a HPLC system. Using a C18-Nova Pak reverse phase column, we were able to separate, identify and quantify the substrate, products and their possible catabolites and UV-detectable inhibitors. A complete separation and quantitation of metabolites was accomplished within 16-18 minutes. However, rapid and simple HPLC runs were also developed which can be routinely used to determine adenosine levels within 3-4 minutes, using a single solvent HPLC system. This procedure is extremely reproducible and very reliable as demonstrated in assaying for 5'-nucleotidase, adenylate deaminase, adenosine deaminase and adenylosuccinate lyase activities.

Myoadenylate deaminase deficiency: primary and secondary types.

Myoadenylate deaminase deficiency, the most common of the known enzyme deficits of muscle, appears to occur in two forms. The primary type seems to be inherited as a complete gene block in an autosomal recessive pattern. Although occasionally diagnosed in infancy, when muscle biopsy is performed on a hypotonic but normoreflexic child, the deficiency is usually not symptomatic until adult or middle age, when muscle cramping and exercise intolerance develop. The skeletal muscle isozyme is immunologically, and presumably genetically, unique, and these patients have normal levels of adenylate deaminase in their other cells and tissues. A presumptive diagnosis can usually be made by an ischemic forearm exercise test, which shows a negligible increase in blood ammonia, despite a normal rise in lactate. Despite the absence of more than 99% of normal adenylate deaminase activity, the muscle biopsy shows no anatomic pathology, and other enzymes are at normal levels. These patients do not suffer progressive disease, and should be reassured and encouraged to maintain physical activity. The heterozygous state is probably asymptomatic, except, perhaps, on extreme exercise, but may be associated with an increased incidence of malignant hyperthermia susceptibility. Since the gene defect is not rare, it is not surprising that some cases of the deficiency will be coincidentally associated with other neuromuscular disease. However, there is also a secondary form of myoadenylate deaminase deficiency, consequent to muscle damage from other disease. In this form, the residual activity is higher (1-10% of normal), may present rare foci of positive stain in the section, and reacts normally with antibody to the muscle isozyme. Other muscle enzymes are also depleted, although not as severely, and the prognosis in such cases is dictated by the primary disease. Since the heterozygous state is common, these patients might have been carriers whose adenylate deaminase levels have been lowered to the deficient category by the advent of other neuromuscular disease.

Amino-acid metabolism enzyme activities in the liver, intestine and yolk sac membrane of developing domestic fowl

To contribute to our understanding of nitrogen metabolism in the developing chick we have studied in liver, intestine and yolk sac membrane the ontogeny of both aspartate- and alanine transaminases, glutamate dehydrogenase, adenylate deaminase, glutamine synthetase and xanthine dehydrogenase activities. Liver enzyme activities were much higher than those of the same enzymes in intestine and yolk sac membrane, the latter having the lowest activities. In the liver, both alanine transaminase and glutamate dehydrogenase increased their activity just before hatching, xanthine dehydrogenase and glutamine synthetase develop their highest activity just after hatching, while aspartate transaminase and adenylate deaminase attained the highest levels just with adulthood. From the pattern of enzyme activity in yolk sac membrane and intestine it can be inferred that after hatching, the amino-acid metabolism in these tissues is considerably enhanced, with higher production of ammonia from amino acids, as indicated by the rise in adenylate deaminase, as well as increased potentiality in production of both alanine and glutamine. It can be concluded that hatching coincides with a deep change of pace in amino-acid metabolism in the organs studied fully comparable with that observed in Mammals at the end of lactation, with the difference that the adaptation to the new diet in the case of the chick is much more sudden than weaning is for the rat.

Myoadenylate deaminase deficiency: Inherited and acquired forms

Myoadenylate deaminase deficiency, the most common of the known enzyme deficits of muscle, appears to occur in two forms. The primary type seems to be inherited as a complete gene block in an autosomal recessive pattern. Although occasionally diagnosed in infancy, when muscle biopsy is performed on a hypotonic but normoreflexic child, the deficiency is usually not symptomatic until adult or middle age, when muscle cramping and exercise intolerance develop. The skeletal muscle isozyme is immunologically, and presumably genetically, unique, and these patients have normal levels of adenylate deaminase in their other cells and tissues. A presumptive diagnosis can usually be made by an ischemic forearm exercise test, which shows a negligible increase in blood ammonia, despite a normal rise in lactate. Despite the absence of more than 99% of normal adenylate deaminase activity, the muscle biopsy shows no anatomic pathology, and other enzymes are at normal levels. These patients do not suffer progressive disease, and should be reassured, and encouraged to maintain physical activity. The heterozygous state is probably asymptomatic, except, perhaps, on extreme exercise, but may be associated with an increased incidence of malignant hyperthermia susceptibility. Since the gene defect is not rare, it is not surprising that some cases of the deficiency will be coincidentally associated with other neuromuscular disease. However, there is also a secondary form of myoadenylate deaminase deficiency, consequent to muscle damage from other disease. In this form, the residual activity is higher (1–10% of normal), may present rare foci of positive stain in the section, and reacts normally with antibody to the muscle isozyme. Other muscle enzymes are also depleted, although not as severely, and the prognosis in such cases is dictated by the primary disease. Since the heterozygous state is common, these patients might have been carriers, whose adenylate deaminase levels have been lowered to the deficient category by the advent of other neuromuscular disease.

Intermediary purine-metabolizing enzymes from the cytosol of Dictyostelium discoideum monitored by high-performance liquid chromatography

The use of high-performance liquid chromatography to identify and quantitate five purine-metabolizing enzymes from a partially purified subcellular fraction of the eucaroytic microorganism Dictyostelium discoideum is described. All HPLC separations were carried out in an isocratic manner using reverse-phase C 18 as the stationary phase. The mobile phase consisted of a phosphate buffer with either methanol or acetonitrile as cosolvent, and optimal separation conditions were attained by varying the organic concentration or the pH of the buffer or by employing pairedion chromatographic techniques. Substrates and products were detected at either 254 nm for the purines or 295 nm for the formycin analogs. An adenosine kinase activity was identified, and it was demonstrated that formyin A (FoA) could be substituted for adenosine as the phosphate acceptor, yielding FoAMP as the product. With FoA as the substrate an apparent K m of 18.2 μ m and an apparent V max of 32.4 mmol min −1 mg −1 were observed for the activity. A purine nucleoside phosphorylase activity was found to cleave adenosine to adenine and ribosylphosphate. FoA was not found to be a substrate for this activity due to the unusual formycin C-glycosyl bond which was not hydrolyzed by enzymes or chemically with either HCl or NaOH. An adenylate deaminase activity was found to be present in the cytosolic S-100 of cells harvested during the onset of development, and this deaminase activity was greatly stimulated by ATP. With FoAMP as the substrate, an apparent K m of 236 μ m and V max of 2.78 μmol min −1 mg −1 were observed. The deamination of FoAMP could be inhibited by the addition of the natural substrate AMP. An apparent K i value of 136 μ m was determined from initial rate data. An adenylosuccinate synthetase activity was observed to have a K m value for GTP, IMP, and aspartic acid of 23, 34, and 714 μ m, respectively. The formycin analog FoIMP was not a substrate with this activity but was a competitive inhibitor of IMP. Finally hypoxanthine-guanine phosphoribosyltransferase was found to have K m and V max values for hypoxanthine of 55.5 μ m and 34.3 nmol −1 min −1 mg 1. When guanine was used as the substrate, the rate of nucleotide formation was 50% that with hypoxanthine as the substrate. The advantages of using HPLC to examine the interconnecting activities of a multienzyme complex in subcellular fractions are discussed, including the increased sensitivity obtained by using formycin analogs in the assay procedures. The methodology is extended to interpreting a series of enzyme reactions in which the product of the first was the substrate for the second.

Ammonia Release by Chum Salmon Eggs at the Initiation of Their Embryonic Development: (ammonia release/egg activation/adenylate deaminase/alkalization/salmon egg).

We have found that inseminated eggs of the chum salmon, Oncorhynchus keta, actively release alkaline substances when they have been stimulated by deionized water; the pH of the egg suspension prepared in the non-buffered Salmon-Ringer solution shifts within at least 1.3 pH units. The nesslerization method and an ammonia gas sensitive electrode measurement reveal that the alkalization of the suspension is partly due to the efflux of ammonia from the stimulated eggs. The increase in the intracellular ammonia level leading to the alkalization of ooplasm may occur at an early stage of the embryonic development. Since the ammonia release is observed also in the eggs stimulated parthenogenetically by deionized water, it can be inferred that the stimulation of the egg triggers a reaction which increases the ammonia level in the ooplasm. The environmental medium of stimulated eggs contains fairly large amounts of organic compounds. Its UV spectrum shows a peak at 249 nm; the position of the absorption maximum corresponds to that of inosine monophosphate (IMP). This fact suggests that IMP is one of the components of the organic compounds. The presence of IMP in the medium is also shown by thin layer chromatography. The medium contains monosaccharides and peptides. We have explained these results by postulating that an abrupt change in the activity of adenylate deaminase which catalyzes the conversion of adenosine monophosphate to ammonia and IMP occurs at the initiation of the embryonic development in the chum salmon egg.