Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

Purine metabolizing enzymes of Plasmodium lophurae and its host cell, the duckling (Anas domesticus) erythrocyte

Adenosine kinase, adenosine deaminase, hypoxanthine phosphoribosyltransferase, inosine-nucleoside phosphorylase, 5'-AMP deaminase and 5'-IMP nucleotidase were identified in cell-free extracts of duckling erythrocytes; no evidence for 5'-AMP nucleotidase and xanthine oxidase activity was found. The Km values for the duckling red cell enzymes were similar to those reported for human erythrocytes. Plasmodium lophurae extracts demonstrated similar enzyme activities except for 5'-AMP deaminase and 5'-IMP nucleotidase which were absent. It is proposed that during infection erythrocytic AMP is catabolized to IMP, inosine and hypoxanthine; the hypoxanthine is taken up by the plasmodium, utilized to form IMP, and this in turn is converted into adenine and guanine nucleotides.     

Enzymes of purine and pyrimidine metabolism from the human malaria parasite, Plasmodium falciparum

Plasmodium falciparum trophozoites were isolated by mechanical rupture of infected human erythrocytes followed by a series of differential centrifugation steps. After lysis with sonication, the 100 000 x g supernatant of parasites and uninfected host cells was used to determine the specific activities of a number of enzymes involved in purine and pyrimidine metabolism. P. falciparum possessed the purine salvage enzymes: adenosine deaminase, purine nucleoside phosphorylase, hypoxanthine-guanine phosphoribosyltransferase (PRTase), xanthine PRTase, adenine PRTase, adenosine kinase. The last two enzymes, however, were present at much lower activity levels. Hypoxanthine was converted (presumably via IMP) into adenine and guanine nucleotides only in the presence both of supernatant and membrane fractions of P. falciparum. Two enzymes involved in the de novo synthesis of pyrimidines, orotic acid PRTase, and orotidine 5'-phosphate decarboxylase, were present in parasite extracts as were the enzymes for pyrimidine nucleotide phosphorylation: UMP-CMP kinase, dTMP kinase, nucleoside diphosphate kinase. Xanthine oxidase, CTP synthetase, cytidine deaminase and several kinases for the salvage of pyrimidine nucleosides were not detected in the parasites. Both phosphoribosyl pyrophosphate synthetase and uracil PRTase were present but at low activity levels. Human erythrocytes displayed similar but not identical enzyme patterns. Enzyme specific activities, however, were generally much lower than those of the corresponding parasite enzymes.     

Cloning and characterization of guanine deaminase from mouse and rat brain.

A search for genes differentially expressed in the rat striatum revealed a gene fragment with a ventral to dorsal striatal expression pattern. The sequence of the fragment was used to isolate mouse and rat clones that upon sequencing were identified as homologous to human guanine deaminase. Here we report the distribution of guanine deaminase in the rodent brain. In situ hybridization localization of the encoding mRNA showed a distribution primarily in forebrain areas including cortical pyramidal neurons, ventral striatal medium spiny neurons, hippocampal pyramidal neurons in CA3-CA1 and granule cells in the dentate gyrus, and neurons of the amygdala. Immunohistochemistry using antibodies raised against peptide fragments derived from the guanine deaminase protein sequence showed localization of guanine deaminase in areas predicted by the mRNA distribution. In addition to immunolabeling of neurons in the cerebral cortex, hippocampus, striatum and amygdala there was also labeling in the terminal fields of these neurons including the thalamus, globus pallidum and substantia nigra. A functional histochemical assay that demonstrates the site of guanine deamination shows guanine deaminase activity in a pattern that matched the immunohistochemical localization. The cellular distribution of guanine deaminase to distal areas of the cell including terminals and dendrites was additionally demonstrated by the expression of recombinant guanine deaminase in transformed cortical neurons in culture.In summary we have described the isolation and characterization of mouse and rat guanine deaminase. The expression of guanine deaminase is primarily restricted to forebrain neurons. A histochemical assay was used to localize guanine deaminase activity to the dendrites and axons of neurons expressing guanine deaminase.     

Deamination of adenosine by extracts of Penicillium politans NRC-510

Cell-free extracts of nitrate-grown Penicillium politans NRC-510 could catalyze the hydrolytic deamination of adenosine to inosine maximally at pH 6.0 and 45 degrees C. However the same extracts could not catalyze the N-glycosidic bond cleavage of adenosine at pH 4.0, 6.0 and 8.0. Incubation of the extracts at 55 degrees C for 30 minutes caused about 31% loss in activity whereas incubation of the extracts at 60 degrees C for 15 minutes caused a complete loss of enzyme activity. Results indicated the absence of the involvement of sulfhydryl groups in the catalytic site of adenosine deaminase. The enzyme is inhibited by ethylene diamine tetraacetate indicating that adenosine deaminase is a metalloenzyme. MnCl2 and MgCl2 had a remarkable activating effect, whereas HgCl2, CaCl2 and ZnSO4 showed an inhibitory effect on enzyme activity. Dialyzing the extracts for 24 hours significantly increase deaminase activity by about 33%. The apparent K(m) value was calculated for adenosine and found to be 3.63 x 10(-3) M, which indicates high affinity of adenosine deaminase for its substrate adenosine.     

Hydrolytic cleavage of purine ribonucleosides in Aspergillus phoenicis

Cell-free extracts of nitrate-grown Aspergillus phoenicis could catalyze the hydrolytic cleavage of the N-glycosidic bond of inosine, guanosine and adenosine to the corresponding base and ribose by the nucleoside hydrolase. No evidence was obtained concerning the hydrolytic degradation of N-glycosidic bond of pyrimidine ribonucleosides namely cytidine and uridine by the same extracts. Optimum pH and temperature for adenosine, guanosine and inosine hydrolysis were the same at pH 3.5 and 55 degrees C, respectively. Citrate buffer showed the highest hydrolase activity when compared to the analogous activity obtained with the other buffers used. The rate of hydrolysis of the three nucleosides was in the order inosine > guanosine > adenosine. Incubation of extracts at 55 degrees C for 15 minutes caused about 85%, 75% and 62% loss of activity with adenosine, guanosine and inosine respectively. Dialyzing the extract caused a decrease in enzyme activity. Addition of inorganic arsenate to the reaction mixture (containing adenosine, guanosine or inosine) did not affect the amount of ribose liberated. Addition of EDTA at a concentration of 5 x 10(-3) M caused an inhibition of about 50%, however a complete inhibition for enzyme activity was obtained at 10(-2) M EDTA. MgSO4, CoSO4 and ZnSO4 at a final concentration of 5 x 10(-3) M showed activation of ribonucleoside hydrolase.     

Enzyme analysis of thymic nurse cells

Thymic nurse cells (TNC) were characterized according to their enzyme content. The following enzymes: Lactate dehydrogenase (LDH) isoenzymes, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) were evaluated. When the enzyme profile of the TNC were compared with the one found in the different thymocyte subpopulations, the ADA/PNP ratio was very low and similar to the early thymocytes, whereas the LDH isoenzymes approached the pattern of the cortical thymocytes. This enzyme profile suggests that the enzyme content of the TNC is dependent on the presence of at least two different thymocyte subpopulations, the early thymocytes and the cortical thymocytes.     

Activities of purine nucleotide metabolizing enzymes in human thymocytes and peripheral blood T lymphocytes: in vitro effect of thymic hormones

Adenosine deaminase (ADA, E.C. 3.5.4.4) and purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1) activities are essential for the normal development and function of T lymphocytes. A comparison of the specific activity of these two enzymes and of ecto-5'-nucleotidase (5'-N, E.C. 3.1.3.5) of human thymocytes with that of peripheral T lymphocytes of children and young adults shows that significant differences exist between the activities of ADA and 5'-N in thymocytes and peripheral T cells. ADA activity is six-fold higher in thymocytes than in peripheral T cells whereas 5'-N activity is approximately one-fourth lower in thymocytes than in T lymphocytes. PNP activity is two-fold higher in T cells. The apparent Km and Vmax values for the three enzymes in thymocytes and peripheral T lymphocytes have been determined. The observed differences in enzyme activity are probably not entirely due to differences in the affinity of the enzymes for their substrates. The enzyme activities of a thymoma removed from a patient with myasthenia gravis had intermediate enzyme levels for ADA and PNP, but 5'-N was similar to peripheral T cells. Exposure of thymocytes in short-term culture to the thymic hormones thymopoietin (the pentapeptide TP5), thymosin fraction V or serum thymic factor (FTS) had no significant stimulatory or inhibitory effect on the activities of the three enzymes under our experimental conditions.     

B cells as well as T cells form deoxynucleotides from either deoxyadenosine or deoxyguanosine.

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.     

Purine metabolism in Leishmania donovani amastigotes and promastigotes

Purine metabolism in Leishmania donovani amastigotes was found to be similar to that of promastigotes with the exception of adenosine metabolism. Adenosine kinase activity in amastigotes is approximately 50-fold greater than in promastigotes. Amastigotes deaminate adenosine to inosine through adenosine deaminase, an enzyme not present in promastigotes. Inosine is cleaved to hypoxanthine and phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase. Promastigotes cleave adenosine to adenine and deaminate adenine to hypoxanthine via adenase, an enzyme not present in amastigotes. Hypoxanthine is phosphoribosylated by hypoxanthine-guanine phosphoribosyltransferase.     

Allosteric modification of adenylate deaminase activity initiation of adenosine deaminase activity by potassium ions

Activation of purified adenylate deaminase from the duck myocardium by K⁺ ions is accompanied by a change in the substrate specificity of the enzyme and appearance of ability to deaminate adenosine and adenine. Adenosine deaminase activity appears with K⁺ in a concentration exhibiting maximal stimulating effect (0.15 M) and it increases with an increase in the K⁺ concentration, parallel with a decrease in Hill's coefficient. It can be concluded from the pH dependence, the character of inhibition by phosphate, and the effect of cations of the alkali metals that deamination of adenosine takes place at natural combining sites of adenylate deaminase, the conformation of which is modified by the activator.Laboratory of Biochemistry of Amines and Other Nitrogenous Bases, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 535–537, November, 1978.     

Activities of purine metabolising enzymes in lymphocytes of neonates and young children: correlates with immune function

Depressed activities of the following purine enzymes have been shown to result in immunodeficiencies: adenosine deaminase (ADA), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and purine nucleoside phosphorylase (PNP). These enzymes and adenosine kinase (AK) were measured in cord blood lymphocytes of premature and small-for-gestational age infants since they have partial immunodeficiencies of unknown biochemical etiology which can persist for many years. We also measured these enzymes in 3 infants with various immunodeficiencies. Activities were compared with appropriate matched control groups. The results indicated normal ADA and PNP but significantly depressed AK (P less than 0.05) and HGPRT (P less than 0.001) activities in 10 premature/SGA infants when compared to 35 full-term normal infants. In the 3 immunodeficient children the results were as follows: Child 1 had a 2- to 3-fold decrease in ADA with normal PNP and AK activities; Child 2 had a 2- to 3-fold decrease in AK, 4-fold decrease in HGPRT with normal PNP and ADA activities; Child 3 had confirmed AIDS and a 4-fold decrease in ADA, 6-fold decrease in HGPRT with normal PNP activity. The possible role of these depressed purine enzyme activities found in lymphocytes is discussed in relation to the imparied immunity seen in these infants.     

Is adenosine deaminase involved in adenosine transport?

The hypothesis that adenosine metabolizing enzymes may have a key role in the transport of adenosine is discussed. The enhancement of adenosine transport by inhibitors of adenosine deaminase (the enzyme which deaminates adenosine to inosine) and the ecto-localization of adenosine deaminase suggest a contribution of the enzyme in taking up nucleosides. Two possible mechanisms are suggested: 1) transport and deamination of adenosine as a coupled process, or 2) uptake of inosine after cleavage of adenosine by ecto-adenosine deaminase. In both cases, the so-called adenosine deaminase binding protein which is a membrane protein could be the real nucleoside transporter. This behaviour of adenosine deaminase as an ectoenzyme anchored to a membrane protein remembers the behaviour of periplasmic binding proteins of bacteria. Thus, adenosine deaminase as well as, for instance, adenosine kinase would be a kind of 'periplasmic proteins' of eukaryotic cells. The function of adenosine deaminase and adenosine kinase would then be to take adenosine and give it to the true transporters.     

Catabolism of L-phenylalanine and L-tyrosine by Rhodobacter sphaeroides OU5 occurs through 3,4-dihydroxyphenylalanine

Rhodobacter sphaeroides OU5 utilized l-phenylalanine as sole source of nitrogen for growth. The metabolites of l-phenylalanine catabolism, i.e. 4-hydroxy phenylalanine (l-tyrosine), 3,4-dihydroxyphenylalanine (DOPA), 3,4-dihydroxyphenyl-pyruvic acid (DOPP), 3,4-dihydroxyphenyllactic acid (DOPLA), 3,4-dihydroxyphenyl-acetic acid (DOPAc) and 3,4-dihydroxybenzoic acid (PC), were identified using liquid chromatography-mass spectroscopy (LC-MS). With 2-oxoglutarate as an amino acceptor, DOPA aminotransferase activity was observed with cell-free extracts and the product DOPP was confirmed through mass analysis. Reductive deamination of DOPA also occurred in the absence of 2-oxoglutarate, whose products were 3,4-dihydroxyphenylpropionic acid (DPPA) and ammonia. The enzyme DOPA-reductive deaminase (DOPARDA) was purified to its homogeneity and characterized. DOPARDA has an obligate requirement for NADH and is functional at low concentrations of the substrate (<150 microM). The molecular mass of the purified enzyme was approximately 274kD and the enzyme could be a heterotetramer of 110, 82, 43 and 39kD subunits as determined by SDS-PAGE.     

Purine and pyrimidine metabolizing activities in Trichomonas vaginalis extracts

Sixty-one purine and pyrimidine metabolizing activities were assayed in extracts of Trichomonas vaginalis. Of these, 43 were detected and quantitated. The only phosphoribosyltransfer activity observed was with uracil. No such activity was observed with adenine, guanine, hypoxanthine, xanthine or orotic acid. The rate of nucleoside cleavage was increased dramatically by the addition of inorganic phosphate. In addition, the extracts could catalyze the synthesis of ribonucleosides from the bases adenine, hypoxanthine, guanine and uracil but not cytosine, thymine or orotic acid, in the presence of ribose 1-phosphate. These data suggest that T. vaginalis contains primarily nucleoside phosphorylases instead of nucleoside hydrolases. Adenosine, deoxyadenosine, guanosine, deoxyguanosine, inosine, uridine, thymidine and GMP were phosphorylated in the presence of ATP. No nucleoside phosphotransferase activity was detected. Deamination of guanine, adenosine, deoxyadenosine, cytidine and deoxycytidine but not adenine was observed. These data suggest that salvage of adenine and guanine for ribonucleotide synthesis in T. vaginalis occurs via a phosphorylase/kinase pathway instead of through a phosphoribosyltransferase pathway which predominates in mammalian cells. In contrast, the pyrimidine base uracil can be converted to UMP via both a phosphoribosyltransferase or a phosphorylase/kinase pathway, analogous to that in mammals.     

Molecular Modelling of the Biosynthesis of the Rna-Editing Enzyme Apobec-1, Responsible for Generating the Alternative forms of Apolipoprotein B

We discovered in 1987 that the shorter form of apolipoprotein B (B48) synthesized in the intestine is due to the action, previously unrecognized in mammalian cells, of an mRNA-editing process, and more recently we demonstrated that this was due to a specific enzyme (APOBEC-1) with cytidine deaminase activity. We show here, by sequence alignment, molecular modelling and mutagenesis, that APOBEC-1 is a cytidine deaminase, responsible for editing apoB mRNA, and that is related in crystal structure to the cytidine deaminase of Escherichia coli (ECCDA). The two enzymes are both homodimers with composite active sites formed with loops from each monomer. In the sequence of APOBEC-1, three gaps compared with ECCDA match the size and contour of the minimal RNA substrate. We propose a model in which the asymmetric binding of one active site to the substrate cytidine which is positioned by the downstream binding of the product uridine and that this helps to target the other active site for deamination.     

Purine‐related metabolites and their converting enzymes are altered in frontal,parietal and temporal cortex at early stages of Alzheimer's disease pathology

Adenosine, hypoxanthine, xanthine, guanosine and inosine levels were assessed by HPLC, and the activity of related enzymes 5′‐nucleotidase (5′‐NT), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) measured in frontal (FC), parietal (PC) and temporal (TC) cortices at different stages of disease progression in Alzheimer''s disease (AD) and in age‐matched controls. Significantly decreased levels of adenosine, guanosine, hypoxanthine and xanthine, and apparently less inosine, are found in FC from the early stages of AD; PC and TC show an opposing pattern, as adenosine, guanosine and inosine are significantly increased at least at determinate stages of AD whereas hypoxanthine and xanthine levels remain unaltered. 5′‐NT is reduced in membranes and cytosol in FC mainly at early stages but not in PC, and only at advanced stages in cytosol in TC. ADA activity is decreased in AD when considered as a whole but increased at early stages in TC. Finally, PNP activity is increased only in TC at early stages. Purine metabolism alterations occur at early stages of AD independently of neurofibrillary tangles and β‐amyloid plaques. Alterations are stage dependent and region dependent, the latter showing opposite patterns in FC compared with PC and TC. Adenosine is the most affected of the assessed purines.     

Computer simulation of ischemic rat heart purine metabolism. I. Model construction.

A model is proposed for the partial depletion of the adenine nucleotide pool in the ischemic perfused rat heart which involves seven enzymes: adenylate cyclase, 3',5'-cyclic AMP phosphodiesterase, 5'-nucleotidase, adenosine kinase, adenosine deaminase, purine nucleoside phosphorylase, and inorganic pyrophosphatase. The computer implementation of this model is in terms of rate laws, several of which were obtained by a systematic least-squares fitting procedure. Depletion of the adenine nucleotide pool is initiated by the release of endogenous noradrenaline into the interstitial fluid, which results from a fall in tissue PO2, and the subsequent activation of adenylate cyclase. In this model the substrate for 5'-nucleotidase is a membrane-bound AMP pool formed by hydrolysis of extracellular fluid and functions as a vasodilator; excess adenosine is incorporated into the tissue by a "permease" with Michaelis-Menten kinetics and converted to AMP, inosine, and hypoxanthine. Alternative mechanisms, such as the deamination of AMP by adenylate deaminase and conversion of AMP to adenine by AMP pyrophosphorylase, were rejected primarily on qualitative biochemical grounds.     

Microassay for blood adenosine deaminase: study of the role of the enzyme in the ammoniagenesis of the blood sample

A microassay for adenosine deaminase is elaborated. It is based on the colorimetric measurement of small amounts of ammonia raised during adenosine deamination and separated by continuous flow dialysis. The analytical qualities of the microassay are reported. The enzymic activity concentration in whole arterial blood of 30 control subjects is 2.54 +/- 1,68 micro katal/l (mean +/- 2ET). It is slightly lower in venous blood. The values are higher in man than in women. 92 p. cent of the enzymic activity of whole blood are due to the erythrocytes. The distribution of the arterial enzymic activity concentrations is not significantly different in a group of 85 cirrhotic patients when compared to the control group. No tight correlation can be found between blood adenosine deaminase concentrations and either blood ammonia nor shed blood ammoniagenesis. Inhibitors of adenosine deaminase have little effects on the in vitro ammoniagenesis.     

Myoadenylate deaminase deficiency

Summary This report concerns two unrelated males; one had sarcoidosis, sarcoid myopathy and muscle weakness, and the other had exercise-induced weakness and myalgia. Both patients had a lack of ammonia rise in their serum after an ischemic work test, minimal histochemical activity of myoadenylate deaminase in repeated muscle biopsies, and less than 5% of normal biochemical activity of myoadenylate deaminase in their skeletal muscles. These three criteria establish primary myoadenylate deaminase deficiency as a separate primary metabolic muscle disease which merits differential diagnostic consideration when patients complain of muscle weakness and cramps.Abbreviations ACE angiotensin converting enzyme - CK creatine kinase - MAD myoadenylate deaminase - MADD myoadenylate deaminase deficiency