Salvage Of Purine Bases Research Articles

Molecular and functional analysis of hypoxanthine‐guanine phosphoribosyltransferase from Arabidopsis thaliana

Hypoxanthine-guanine phosphoribosyltransferase (HGPT) occurs in both eukaryotic and prokaryotic organisms. However, the molecular and functional properties of plant HGPT are not well understood. In this study, it was found that the putative HGPT proteins from dicot and monocot plant species exhibited significant identities to their homologs from other cellular organisms. Ectopic expression of the HGPTs from Arabidopsis, soybean or wheat complemented HGPT deficiency in the hpt1 mutant of Saccharomyces cerevisiae. Recombinant Arabidopsis HGPT (AtHGPT) catalyzed both forward and reverse reactions in in vitro biochemical assays. The relative catalytic efficiency for the synthesis of guanosine monophosphate (GMP) was significantly greater than that for the production of guanine from GMP. Further investigations led to identification of the candidate residues that may form the pyrophosphate (PPi) binding loop in AtHGPT. AtHGPT expression level was dynamically regulated in Arabidopsis organs and during leaf development and senescence and seed germination. AtHGPT knockout mutant germinated more slowly than wild type control, whereas its overexpression mutant exhibited accelerated germination. Collectively, the data suggest that functional HGPTs are expressed in higher plants. In Arabidopsis, HGPT plays an active role in the salvage of purine bases and its activity is required for efficient seed germination.

A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.

Uridine preserves ATP during hypoxic perfusion of the rat heart

Background: The pyrimidine precursor, orotic acid, by minimising ischaemia-induced ATP loss, improves the functional performance of recently infarcted hearts that have been subjected to global ischaemia. However, we have also previously shown that orotic acid is not directly active in the heart but is preferentially taken up by the liver where it is metabolised to uridine. Aim: To investigate whether uridine itself can minimise hypoxia-induced ATP loss. Methods: Isolated Langendorff-mode perfused rat hearts were subjected to 4 protocols after 20 min normoxic stabilisation: normoxia for 30 min (n=12); hypoxia for 30 min (n=12); hypoxia in the presence of 17μM uridine for 30 min (n=12); and [U- 14C]-uridine added directly to the hypoxic perfusate reservoir just prior to 30 min hypoxia (n=4). [U- 14C]-uridine was used to assess the contribution of radiolabel to adenosine formation from adenine nucleotide hydrolysis. Coronary effluent was collected and hearts were freeze-clamped for metabolite assay. Results: Hypoxia reduced ATP, from 21.1±1.1 to 4.1±0.6 μmol/g dry weight (p<0.05), and reduced total adenine nucleotides (TAN) from 30±1.2 to 10.2±0.9 μmol/g dry weight (p<0.05). Uridine during hypoxia increased myocardial ATP by 94% to 8±0.9 μmol/g dry weight and TAN by 50% to 15.3±1.1 pmol/g dry weight (p<0.05). Uridine plus hypoxia increased total lactate release by 52% from 768.1± 86 μmol/g dry weight to 1148.4 ±146 μmol/g dry weight compared with hypoxia alone (p<0.05). Although the salvage of purine bases did occur, it was calculated that less than 0.01% of labelled ribose was transferred for salvage of purines. Conclusion: In the present experimental model, uridine protects the hypoxic heart by predominantly enhancing glycolytic energy production.

The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties

Tritrichomonas foetus, an anaerobic, flagellated protozoan parasite, is incapable of de novo purine nucleotide synthesis, and depends primarily on the salvage of purine bases from the host. The hypoxanthine-guanine-xanthine phosphoribosyl-transferase (HGXPRTase) from this organism has been purified to homogeneity by ammonium sulfate precipitation and Sephacryl-HR100 gel filtration, followed by anion exchange FPLC. Hypoxanthine, guanine and xanthine phosphoribosyl-transferase activities co-eluted in all the purification steps, suggesting that they are associated with the same enzyme protein. The molecular mass of the native protein, as estimated by gel filtration, is 24 kDa. The molecular mass estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is also 24 kDa. Non-denaturing polyacrylamide gel electrophoresis of the purified protein, followed by activity staining with either [ 14C]hypoxanthine, [ 14C]guanine or [ 14C]xanthine, also demonstrates that the enzyme is a monomer of 24 kDa. This monomeric structure is distinctive from all the other reported PRTases which are either dimers or tetramers. Furthermore, unlike the mammalian HGPRTase, which is heat stable, the T. foetus enzyme is heat labile. Kinetic studies with the purified T. foetus HGXPRTase showed that the apparent Kms for hypoxanthine, guanine and xanthine were 4.1 μM, 3.8 μM and 52.4 μM respectively. This recognition of xanthine as a substrate by the parasite enzyme with only about a 10-fold higher Km value than those for hypoxanthine and guanine distinguishes it from the mammalian HGPRTase, which cannot use xanthine as a substrate, as well as the HGXPRTases of Eimeria tenella and Plasmodiumfalciparum, which are dimers, with xanthine about 100-times less proficient as a substrate. T. foetus HGXPRTase is thus a unique enzyme with opportunity for specific inhibitor design.

Uric acid metabolism in humans.

U RIG ACID, the end product of purine degradation in humans, gains clinical significance during states of increased production, decreased elimination, or increased renal excretion. Under these conditions urate can be deposited in tissues, or uric acid can precipitate in urine to cause uric acid nephrolithiasis. Disorders associated with abnormal uric acid homeostasis include Lesch-Nyhan syndrome, an inherited enzyme deficiency, gout, renal insufficiency, and increased cellular breakdown as in tumor lysis. This article focuses on the sources, production, and elimination of uric acid in man. Uric acid is a weak acid with a pKal of 5.75 due to a proton loss at position 9. l At the physiologic pH range of human serum, uric acid is a monovalent ion, but in urine, which has a more acidic pH range, it is mostly excreted as the free acid. Thus, when uric acid is deposited in tissues, as seen in tophaceous gout, the deposits are mostly monosodium urate utilizing the most abundant cation, and uric acid renal stones are largely free acid.* Uric acid can also lose a second proton at position 3 with a pKa, of 10.3.3 The solubility of uric acid, like the ionization, is pH-dependent, increasing as the pH and urate formation increase.4 In urine, at a pH of 5 .O, uric acid is soluble to 15 mg/dL and increases with increasing pH to 1,520 mg/dL at a pH of 8.0.5 From this information, it is obvious to see why urine alkalinization is an important therapy to prevent or dissolve uric acid kidney stones. To fully understand the metabolism of uric acid, it is important to begin with the source of uric acid, purines. There are three sources of purines: (1) dietary intake of foods containing purine bases, (2) de novo endogenous synthesis from nonpurine precursors, and (3) salvage of purine bases from endogenous catabolism. The major source of dietary purine is protein. The average diet contains 70 to 100 g/d of proteins.6 Hyperuricemia is induced by a high dietary intake of meats containing nucleic acid and mononucleotides.7.8 Zollner reported the intestinal absorption of mononucleotides to be complete, and the absorption of ribonucleotides to be only

13 INHERITED SUPERACTIVITY OF PRPP SYNTHETASE (PS): ASSOCIATION OF PURINE OVERPRODUCTION AND SENSORINEURAL DEAFNESS

In fibroblasts from an 8 year-old male (VRG) with tophaceous gout, uric acid overproduction, and sensorineural deafness, and from his gouty mother, superactivity of PS due to purine nucleotide inhibitor-resistance was found. Compared with normal cells, VRG fibroblasts showed increases in the following: PRPP concentration (3-fold) and generation (2-fold); purine synthesis de novo (2.5-fold); purine base salvage (2.6-fold); ATP and GTP concentrations (1.4-fold); and excretion of newly synthesized purines (5-fold). Derangements in PS and in PRPP and purine synthesis were attenuated in cells from the mother, suggesting that VRG is a hemizygote for an X chromosome-linked defect and his mother is a heterozygote. Comparison was made of affected males in 8 families with PS superactivity. VRG was intermediate in range of expression of PS superactivity and in severity of the enzyme defect as measured by the degree of aberration of PRPP and purine nucleotide synthesis in fibroblasts. Metabolic abnormalities were more severe in VRG than in most patients (in whom expression is limited to early adult-onset gout with uric acid overproduction), but less severe than in 2 patients with more complex defects in PS associated with uric acid over-production and neurodevelopmental abnormalities, including deafness in hemizygous male children and heterozygous women. Within the spectrum of defects resulting in PS superactivity, certain derangements may be causally related to neurological impairment, most commonly sensorineural deafness.

Superactivity of human phosphoribosyl pyrophosphate synthetase due to altered regulation by nucleotide inhibitors and inorganic phosphate

Phosphoribosyl pyrophosphate ( PPRib P) synthetase activity was studied in cultured fibroblasts and lymphoblasts from a male child (patient 2-A) in whom inherited purine nucleotide and uric acid overproduction are accompanied by neurological deficits. Chromatographed or partially purified preparations of the child's enzyme showed 5–6-fold increased inhibitory constants ( I 0.5) for the noncompetitive inhibitors GDP and 6-methylthioinosine monophosphate but normal responsiveness to the competitive inhibitors ADP and 2,3-diphosphoglycerate. Activation of the PPRib P synthetase of patient 2-A by P i was also abnormal with 3–4-fold reduced apparent K D values for P i. Superactivity of the PPRib P synthetase of this child thus appeared to result from a combination of regulatory defects; selective resistance to noncompetitive inhibitors and increased responsiveness to P i activation. Selective growth of the patient's fibroblasts in medium containing 6-methylthioinosine confirmed the functional significance of the in vitro inhibitor resistance of the aberrant enzyme. Fibroblasts and lymphoblasts derived from patient 2-A showed increased concentrations and rates of generation of PPRib P as well as increased rates of the pathways of purine base salvage and purine nucleotide synthesis de novo. The magnitudes of these increases in the child's cells exceeded those in cells with catalytically superactive PPRib P synthetases. These alterations as well as the in vitro kinetic abnormalities in the patient 2-A enzyme were expressed to a reduced degree in fibroblasts from the child's affected mother, supporting the proposal that this woman is a heterozygous carrier for X-linked enzyme superactivity.

PURINE NUCLEOTIDE SYNTHESIS IN CULTURED RAT EMBRYOS UNDERGOING ORGANOGENESIS: 174

The cultured rat embryo undergoing organogenesis (9.5-11.5 days of gestation) together with its associated yolk sac synthesize purine nucleotides via the de novo synthetic pathway. Although both the embryo and its yolk sac contain significant levels of the purine base salvage enzymes adenine and hypoxanthine phosphoribosyltransferase, the culture medium that consists largely of rat serum contains high activity levels of purine catabolic enzymes. Short-term pulse-chase experiments with adenine and guanine, carried out under virtually serum-free conditions, confirmed that purine base salvage mechanisms were active and that there was no significant net transfer of purines between the embryo and its yolk sac. The 3-carbon atom of serine is the major source of one-carbon units for purine ring synthesis with a significant contribution from the 2-ring carbon atom of tryphtophan. No one-carbon units are derived from glycine, histidine or choline. Most of the embryonic amino acid requirements are supplied by yolk sac mediated proteolysis of the culture medium protein. A high level of embryonic GTP is reflected in a very low ATP/GTP ratio and is presumably related to the relatively undifferentiated state of many of the cells. The reason for the high level of GTP is however purely conjectional.

Purine nucleotide metabolism in resident and activated rat macrophages in vitro

The overall purine metabolism was studied in detail in resident peritoneal macrophages (M phi) and in thioglycolate elicited peritoneal M phi in vitro. The salvage of purine bases (adenine, hypoxanthine and guanine) was active in both M phi populations, whereas purine biosynthesis de novo was low. Purine nucleosides (inosine, guanosine and adenosine) were efficiently degraded to uric acid and only adenosine was directly salvaged into nucleotides. Purine salvage was markedly increased in elicited M phi as compared to resident M phi whereas purine degradation pathways were enhanced only slightly. These results clearly indicate that salvage of purine bases is the main source for purine nucleotide biosynthesis in M phi, but nucleotide catabolism is the predominant pathway.

De novo purine synthesis in cultured rat embryos undergoing organogenesis.

The cultured rat embryo undergoing organogenesis (9.5-11.5 days of gestation) together with its associated yolk sac synthesize purine nucleotides via the de novo synthetic pathway. Although both the embryo and its yolk sac contain significant levels of the purine base salvage enzymes adenine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the culture medium that consists largely of rat serum contains no measurable quantities of salvageable purine bases or nucleosides but high activity levels of purine catabolic enzymes. Short-term pulse-chase experiments with adenine and guanine, carried out under virtually serum-free conditions, confirmed that purine base salvage mechanisms were active and that there was no significant net transfer of purines between the embryo and its yolk sac. A comparison between the specific radioactivities of the [14C]glycine added to the culture medium for the studies of the de novo synthetic pathway and the purine bases in both the cellular nucleotides and the nucleic acids indicated the existence of a large glycine pool, which almost certainly was derived from the degradation of medium serum proteins by the yolk sac. Although there are no clear-cut data available on the in vivo plasma levels of purines that could be potentially utilized to meet the demands of the embryo, it is evident that the de novo pathway is adequately developed to meet these needs.

Adenine Nucleotide Degradation by the Obligate Intracellular Bacterium Rickettsia typhi

Adenosine 5'-triphosphate (ATP) was catabolized by whole cells and cell-free extracts of Rickettsia typhi to adenosine 5'-diphosphate (ADP) and then to adenosine 5'-monophosphate (AMP), the end product of ATP catabolism under the experimental conditions used. The only intermediate of the pathway from ATP to AMP which was identified by thin-layer chromatography and quantitated by the (14)C content was ADP, whereas products such as adenine, adenosine, hypoxanthine, inosine, and inosine 5'-monophosphate were not detected. The enzymes which could be theoretically responsible for the catabolism or the anabolism of AMP were not detected by standard assay procedures. Most importantly, 5'-nucleotidase or nonspecific phosphatase and AMP nucleosidase activities were undetectable under a variety of experimental conditions. Although these two enzymes remove AMP from the adenylate pool in other cells, they are apparently nonfunctional in R. typhi. The biosynthesis of ATP was initiated by adenylate kinase because no adenine phosphoribosyltransferase or adenosine kinase could be detected. Furthermore, AMP was transported intact without prior dephosphorylation. These observations suggest that for R. typhi the in vivo activity of adenine nucleotide interconversion was limited to the nucleotides, with AMP being the end product of ATP catabolism, and that the salvage of purine bases and nucleosides was not an essential feature of purine metabolism. These results elucidate the findings of a previous study which showed that in the absence of glutamate as a source of energy, the adenylate energy charge of resting cells of R. typhi is drastically lowered by the high proportion of AMP.