Adenine phosphoribosyltransferase: A simple spectrophotometric assay and the incidence of mutation in the normal population

The significance of partial deficiency of erythrocyte adenine phosphoribosyltransferase (APRT), reported in a number of subjects with gout, has been investigated by studying its incidence in 700 normal blood donors. Three clearly deficient subjects were found, an incidence not significantly different from that in patients with abnormalities of urate metabolism. A new assay method for APRT is described in which an erythrocyte lysate is incubated with adenine and phosphoribosylpyrophosphate (PRPP) for a given time; both hemoglobin and adenine nucleotide (AMP) are then precipitated with lanthanum phosphate; the change in absorbance of adenine at 260 nm reflects the extent of its conversion to AMP by APRT.This work was supported by the National Health and Medical Research Council of Australia.     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type=Italic>Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to β-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.     

Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866

Visfatin/pre-B cell colony-enhancing factor 1 (PBEF)/nicotinamide phosphoribosyltransferase (NAmPRTase) is a multifunctional protein having phosphoribosyltransferase, cytokine and adipokine activities. Originally isolated as a cytokine promoting the differentiation of B cell precursors, it was recently suggested to act as an insulin analog via the insulin receptor. Here, we describe the first crystal structure of visfatin in three different forms: apo and in complex with either nicotinamide mononucleotide (NMN) or the NAmPRTase inhibitor FK-866 which was developed as an anti-cancer agent, interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. The crystal structures of the complexes with either NMN or FK-866 show that the enzymatic active site of visfatin is optimized for nicotinamide binding and that the nicotinamide-binding site is important for inhibition by FK-866. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin and does not share the binding site with insulin on the insulin receptor. To predict binding sites, the potential interaction patches of visfatin and the L1-CR-L2 domain of insulin receptor were generated and analyzed. Although the relationship between the insulin-mimetic property and the enzymatic function of visfatin has not been clearly established, our structures raise the intriguing possibility that the glucose metabolism and the NAD biosynthesis are linked by visfatin.     

Metabolic properties of an azaguanine-resistant variant of Chinese hamster ovary cells (azarts) with normal levels of hypoxanthine-guanine phosphoribosyltransferase activity

Azarts Chinese hamster ovary cells were 20 to 50 times more resistant to 8-azaguanine and 50 to 10 times more resistant to both 6-thioguanine and 6-mercaptopurine than wild-type cells. Resistance correlated with a failure of azarts cells to incorporate 8-azaguanine into the nucleotide pool and into nucleic acids. The uptake of hypoxanthine and guanine, on the other hand, was about the same in both types of cells and the hypoxanthine-guanine phosphoribosyltransferase of the azarts cells as measured in cell lysates was unaltered both in concentration and kinetic properties with hypoxanthine as well as 8-azaguanine as substrate. Plasma membrane permeability to 8-azaguanine and the regulation of intracellular pH were also not altered in azarts cells and there was no significant degradation of 8-azaguanine or azaguanine nucleotides. We conclude therefore that in azarts cells the phosphoribosylation of 8-azaguanine per se is specifically blocked but that this effect is abolished upon cell lysis.     

Comparison of WNK4 and WNK1 kinase and inhibiting activities

WNK kinases are novel serine/threonine protein kinases. Mutations in two members of the WNK family, WNK1 and WNK4, cause familial hyperkalemic hypertension. These kinases regulate ion transport across diverse epithelia; WNK4 reduces activity of the Na-Cl cotransporter activity and the potassium channel, ROMK, by reducing their appearance at the plasma membrane. We examined the kinase activity of WNK1 and WNK4 in vitro. A glutathione S-transferase (GST) fusion protein of the WNK1 kinse domain phosphorylated itself and a substrate protein, as reported previously. A longer construct, containing the autoinhibitory domain, did not. A GST WNK4 kinase domain construct demonstrated no kinase activity, in vitro or in HEK 293 cells. WNK4 constructs that included a region homologous to the autoinhibitory domain of WNK1 inhibited WNK1 kinase activity. Inhibition by a short WNK4 segment, WNK4 (444-518), was greater than inhibition by WNK4 (444-563). Together, these results suggest that WNK4 must be activated by currently unknown factors to exhibit kinase activity and that WNK4 contains an inhibitory domain that can inhibit the kinase activity of WNK1.     

Organising activities of engrailed, hedgehog, wingless and decapentaplegic in the genital discs of Drosophila melanogaster

 The genes engrailed (en), hedgehog (hh), wingless (wg) and decapentaplegic (dpp) have been shown to play vital organising roles in the development and differentiation of thoracic imaginal discs. We have analysed the roles of these genes in organising the development and differentiation of the genital discs, which are bilaterally symmetrical and possess different primordia, namely, the male and female genital primordia and an anal primordium. Our results suggest that the organising activity of en in genital discs programs the normal development and differentiation of the genital disc by regulating the expression of hh. Hh in turn induces wg and dpp, the genes whose products act as secondary signalling molecules. Moreover, the complementary patterns of wg and dpp expression are essential for the bilateral symmetry and are maintained by mutual repression. Received: 20 April 1998 / Accepted 24 June 1998     

Purification and characterization of Escherichia coli xanthine-guanine phosphoribosyltransferase produced by plasmid pSV2gpt

The enzyme xanthine-guanine phosphoribosyltransferase from scherichia coli cells harboring the plasmid pSV2gpt has been purified 30-fold to near homogeneity by single-step GMP-agarose affinity chromatography. It has a K_m value of 2.5, 42 and 182 μM for the substrates guanine, xanthine and hypoxanthine, respectively, with guanine being the most preferred substrate. The enzyme exhibits a K_m value of 38.5 μM for PRib-PP with guanine as second substrate and of 100 μM when xanthine is used as the second substrate. It is markedly inhibited by 6-thioguanine, GMP and to a lesser extent by some other purine analogues. Thioguanine has been found to be the most potent inhibitor. The subunit molecular weight of xanthine-guanine phosphoribosyltransferase was determined to be 19 000. The in situ activity assay on a nondenaturing polyacrylamide gel electrophoresis gel has indicated that a second E. coli phosphoribosyltransferase preferentially uses hypoxanthine as opposed to guanine as a substrate, and it does not use xanthine.     

Spatial recruitment and activation of the Fes kinase by ezrin promotes HGF-induced cell scattering

The remodeling of epithelial monolayers induced by hepatocyte growth factor (HGF) results in the reorganization of actin cytoskeleton and cellular junctions. We previously showed that the membrane-cytoskeleton linker ezrin plays a major role in HGF-induced morphogenic effects. Here we identified a novel partner of phosphorylated ezrin, the Fes kinase, that acts downstream of ezrin in HGF-mediated cell scattering. We found that Fes interacts directly, through its SH2 domain, with ezrin phosphorylated at tyrosine 477. We show that in epithelial cells, activated Fes localizes either to focal adhesions or cell-cell contacts depending on cell confluency. The recruitment and the activation of Fes to the cell-cell contacts in confluent cells depend on its interaction with ezrin. When this interaction is impaired, Fes remains in focal adhesions and as a consequence the cells show defective spreading and scattering in response to HGF stimulation. Altogether, these results provide a novel mechanism whereby ezrin/Fes interaction at cell-cell contacts plays an essential role in HGF-induced cell scattering and implicates Fes in the cross-talk between cell-cell and cell-matrix adhesion.     

Genetic and biochemical characterization of Corynebacterium glutamicum ATP phosphoribosyltransferase and its three mutants resistant to feedback inhibition by histidine

ATP phosphoribosyltransferase (ATP-PRT) catalyzes the condensation of ATP and PRPP at the first step of histidine biosynthesis and is regulated by a feedback inhibition from product histidine. Here, we report the genetic and biochemical characterization of such an enzyme, HisG_Cg, from Corynebacterium glutamicum, including site-directed mutagenesis of the histidine-binding site for the first time. Gene disruption and complementation experiments showed that HisG_Cg is essential for histidine biosynthesis. HisG_Cg activity was noncompetitively inhibited by histidine and the α-amino group of histidine were found to play an important role for its binding to HisG_Cg. Homology-based modeling predicted that four residues (N215, L231, T235 and A270) in the C-terminal domain of HisG_Cg may affect the histidine inhibition. Mutating these residues in HisG_Cg did not cause significant change in the specific activities of the enzyme but resulted in the generation of mutant ones resistant to histidine inhibition. Our data identified that the mutant N215K/L231F/T235A resists to histidine inhibition the most with 37-fold increase in K_i value. As expected, overexpressing a hisG_Cg gene containing N215K/L231F/T235A mutations in vivo promoted histidine accumulation to a final concentration of 0.15 ± 0.01 mM. Our results demonstrated that the polarity change of electrostatic potential of mutant protein surface prevents histidine from binding to the C-terminal domain of HisG_Cg, resulting in the release of allosteric inhibition. Considering that these residues were highly conserved in ATP-PRTs from different genera of Gram-positive bacteria the mechanism by histidine inhibition as exhibited in Corynebacterium glutamicum probably represents a ubiquitously inhibitory mechanism of ATP-PRTs by histidine.     

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5′ exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R² values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.     

Signal transduction through the T cell receptor is dynamically regulated by balancing kinase and phosphatase activities

Within seconds of T cell receptor engagement, a well-characterized series of tyrosine phosphorylation events mediate cellular activation. It is widely accepted that these initial phosphorylations remain stable until protein tyrosine phosphatases return the cell to its basal level. Based on a model of peripheral blood T cell activation, in which the kinetics of phosphorylation can be modulated, we propose an alternate hypothesis that T cell signaling is highly dynamic. Our results demonstrate that tyrosine phosphorylation and dephosphorylation are occurring co-temporally after T cell receptor cross-linking, regulated by a delicate balance of kinases and phosphatases.     

Determination of intracellular choline levels by an enzymatic assay

A sensitive enzymatic assay for the measurement of intracellular choline is described. The separation of choline from choline-containing phospholipids is accomplished by a minor modification of the Folch technique. The method is based on the specific oxidation of choline by choline oxidase. Phenol and 4-aminoantipyrine in the presence of hydrogen peroxide generated by the oxidation of choline and peroxidase form a red quinone dye which can be detected spectrophotometrically. The assay was useful between 12.5 and 100 nanomoles of choline. The recovery of standard choline in liver homogenates averaged 102 +/- 1.6%. Structurally similar compounds produced minimal interference.     

Discovery of potent and efficacious urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors with reduced CYP2C9 inhibition properties

Potent, reversible inhibition of the cytochrome P450 CYP2C9 isoform was observed in a series of urea-containing nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. This unwanted property was successfully removed from the described inhibitors through a combination of structure-based design and medicinal chemistry activities. An optimized compound which did not inhibit CYP2C9 exhibited potent anti-NAMPT activity (17; BC NAMPT IC₅₀ = 3 nM; A2780 antiproliferative IC₅₀ = 70 nM), good mouse PK properties, and was efficacious in an A2780 mouse xenograft model. The crystal structure of this compound in complex with the NAMPT protein is also described.     

Effect of hydroxyurea on subcellular activities of thymidine kinase in developing and aging rat brain

Hydroxyurea, when injected intraperitoneally at a dose of 1 mg/g body weight, inhibited thymidine kinase activity in developing rat cerebrum (16-day-embryonic) and cerebellum (7-day-postnatal) within a few hours of administration. The inhibition was time-dependent and both cytosolic and mitochondrial thymidine kinases were affected. Under the same conditions, the activities of certain other enzymes concerned with DNA metabolism,viz., DNA polymerase, and acid and alkaline DNases were not inhibited. Further, the addition of hydroxyureain vitro had no effect on the activity of any of the enzymes studied. However, similar treatment given to 2-year-old rat failed to exert any inhibition on either the mitochondrial or soluble thymidine kinase activities in grey and white matter regions of cerebrum and cerebellum. It is inferred that hydroxyurea, apart from its already known effect on ribonucleotide reductase of replicating cells, also affects thymidine kinase.     

Structural and kinetic characterization of quinolinate phosphoribosyltransferase (hQPRTase) from homo sapiens

Human quinolinate phosphoribosyltransferase (EC 2.4.2.19) (hQPRTase) is a member of the type II phosphoribosyltransferase family involved in the catabolism of quinolinic acid (QA). It catalyses the formation of nicotinic acid mononucleotide from quinolinic acid, which involves a phosphoribosyl transfer reaction followed by decarboxylation. hQPRTase has been implicated in a number of neurological conditions and in order to study it further, we have carried out structural and kinetic studies on recombinant hQPRTase. The structure of the fully active enzyme overexpressed in Escherichia coli was solved using multiwavelength methods to a resolution of 2.0 A. hQPRTase has a alpha/beta barrel fold sharing a similar overall structure with the bacterial QPRTases. The active site of hQPRTase is located at an alpha/beta open sandwich structure that serves as a cup for the alpha/beta barrel of the adjacent subunit with a QA binding site consisting of three arginine residues (R102, R138 and R161) and two lysine residues (K139 and K171). Mutation of these residues affected substrate binding or abolished the enzymatic activity. The kinetics of the human enzyme are different to the bacterial enzymes studied, hQPRTase is inhibited competitively and non-competitively by one of its substrates, 5-phosphoribosylpyrophosphate (PRPP). The human enzyme adopts a hexameric arrangement, which places the active sites in close proximity to each other.     

Human TRPM8 and TRPA1 pain channels,including a gene variant with increased sensitivity to agonists (TRPA1 R797T), exhibit differential regulation by SRC-tyrosine kinase inhibitor

TRPM8 (transient receptor potential M8) and TRPA1 (transient receptor potential A1) are cold-temperature-sensitive nociceptors expressed in sensory neurons but their behaviour in neuronal cells is poorly understood. Therefore DNA expression constructs containing human TRPM8 or TRPA1 cDNAs were transfected into HEK (human embryonic kidney cells)-293 or SH-SY5Y neuroblastoma cells and G418 resistant clones analysed for effects of agonists and antagonists on intracellular Ca²⁺ levels. Approximately 51% of HEK-293 and 12% of SH-SY5Y cell clones expressed the transfected TRP channel. TRPM8 and TRPA1 assays were inhibited by probenecid, indicating the need to avoid this agent in TRP channel studies. A double-residue mutation in ICL-1 (intracellular loop-1) of TRPM8 (SV762,763EL, mimicking serine phosphorylation) or one in the C-terminal tail region (FK1045,1046AG, a lysine knockout) retained sensitivity to agonists (WS 12, menthol) and antagonist {AMTB [N-(3-Aminopropyl)-2-[(3-methylphenyl)methoxy]-N-(2-thienylmethyl)benzamide]}. SNP (single nucleotide polymorphism) variants in TRPA1 ICL-1 (R797T, S804N) and TRPA1 fusion protein containing C-terminal (His)₁₀ retained sensitivity to agonists (cinnamaldehyde, allyl-isothiocyanate, carvacrol, eugenol) and antagonists (HC-030031, A967079). One SNP variant, 797T, possessed increased sensitivity to agonists. TRPA1 became repressed in SH-SY5Y clones but was rapidly rescued by Src-family inhibitor PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine]. Conversely, TRPM8 in SH-SY5Y cells was inhibited by PP2. Further studies utilizing SH-SY5Y may identify structural features of TRPA1 and TRPM8 involved in conferring differential post-translational regulation.     

Ecdysone 20-hydroxylation in imaginal wing discs of Pieris brassicae (lepidoptera): Correlations with ecdysone and 20-hydroxyecdysone titers in pupae

Ecdysone 20-hydroxylase activity has been detected in pupal wing discs of Pieris brassicae. This activity is due to an enzyme system located in microsomal fractions. Its apparent Km is 58 nM for ecdysone. The enzyme is inhibited by the reaction product 20-hydroxyecdysone with an apparent Ki of 2.6 μM. Its activity varied during pupal-adult development with a maximum on day 4, when ecdysone levels are the highest in the animal. Although low, the peak activity is sufficient to assure 25% of the conversion of endogenous ecdysone into 20-hydroxyecdysone in pupae. Ecdysone and 20-hydroxyecdysone levels were measured in hemolymph and whole animals; ecdysone appears to be mainly located in hemolymph, whereas 20-hydroxyecdysone seems to be equally distributed between hemolymph and tissues. All these findings are discussed in relation to the roles of ecdysone and 20-hydroxyecdysone during pupal-adult development.     

Changes in protein kinase C and cAMP-dependent kinase in lymphocytes after treatment with 12-O-tetradecanoylphorbol-13-acetate or concanavalin A: quantitation of activities with an in situ gel assay

Primary lymphocytes can be stimulated to proliferate by mitogenic lectins such as concanavalin A (Con A). While the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) alone is not mitogenic for these cells, it can enhance the response to Con A. Previously, protein kinases and phosphorylation have been reported to be important in lymphocyte proliferation. More recently TPA has been found to bind and activate protein kinase C. Therefore, we examined kinase activity in lymphocytes stimulated with the complete mitogen Con A and the comitogen TPA. In order to monitor more than one kinase we used an in situ gel assay and developed the system to compare both protein kinase C and cAMP-dependent kinases. When total cell extracts were assayed in the presence of histone five major bands of activity were detected by autoradiography of the gel. The bands corresponding to protein kinase C and to cAMP-dependent kinases were identified by partial purification of the enzymes, by binding of [20-3H(N)]7-phorbol-12, 13-dibutyrate (3H-PDBU), and by photoaffinity labelling with 8-azidoadenosine-3':5'-cyclic monophosphate (8-N3-[32P]cAMP). Differential extraction of cell lysate allowed comparison of soluble and particulate kinases. We found that when the preparations from either TPA- or Con A-treated lymphocytes were assayed, protein kinase C activity increased three- to four-fold in the particulate fraction within 5 min after treatment. A concurrent decrease of 30-50% occurred in the cytosol. In contrast, cytosolic cAMP-dependent protein kinase II increased 1.4-fold in the same period with Con A. PKI and PKII showed the most significant changes after 24 h of stimulation by Con A when the activity of the holoenzyme decreased to half that of the unstimulated cells. Therefore, although TPA and Con A separately can affect protein kinase C this alone is not sufficient for proliferation to occur.