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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe two retroviral vector-based recombination substrate systems designed to assay for lymphoid VDJ recombinase activity in cultured cells. Both substrates incorporate a constitutive dominant marker gene (the simian virus promoter-driven neo gene) to allow selection of cells that stably integrate the substrate. Both substrates also include a second marker gene that becomes transcriptionally active only when inverted by a site-specific recombination event between flanking immunoglobulin variable-region gene segments. The first vector, similar in structure to previous retrovirus-based recombination substrates, utilizes the bacterial guanine-xanthine phosphoribosyltransferase gene (gpt) as its activatable marker; detection of inversion (VDJ recombinase activity) involves drug selection and Southern blotting analyses. We have used this vector to make a more extensive and quantitative survey of VDJ recombinase activity in B-lineage cell lines than has previously been performed with stable substrates, and we have compared our results with those of other studies that use transient recombination substrates. In the second vector, the activatable gene is the bacterial beta-galactosidase gene (lacZ). Detection for inversional activation of this gene is achieved by a fluorogenic assay, termed FACS-Gal, that detects beta-galactosidase activity in viable cells. The latter assay has the unique advantage of rapidly detecting cells that undergo recombination and also allows viable sorting of cells on the basis of the presence or absence of VDJ recombinase activity. We have used the lacZ vector to rapidly quantitate VDJ recombinase activity in B-lineage cell lines and compared the results with those obtained with the gpt vector. We have also used the lacZ vector to isolate variant pre-B-cell lines with low and high levels of VDJ recombinase activity.
Mol Cell Biol 1990 Apr
PMID:A novel fluorescence-based system for assaying and separating live cells according to VDJ recombinase activity. 232 7

The transcriptional activity of five intracisternal A-particle (IAP) long terminal repeats (LTRs) in mouse embryonal carcinoma PCC3-A/1 cells and in Ltk- cells was determined. We tested the promoter activity of the LTRs by coupling them to the reporter gene chloramphenicol acetyltransferase (CAT) or guanosine-xanthine phosphoribosyltransferase (gpt). Each LTR was tested for promoter function in both the sense (5' to 3') and antisense (3' to 5') orientation preceding the reporter gene. The transcriptional activity of individual IAP gene LTRs varied considerably, and the LTR from IAP81 possessed promoter activity in both directions. The bidirectional activity of the IAP81 LTR confirmed by monitoring Ecogpt expression in stably transfected Ltk- cells, with the initiation sites for sense and antisense transcription being localized to within the IAP81 LTR by S1 nuclease mapping. Deletions of LTR81 show that for normal 5'-to-3' gene transcription (sense direction), the 3'U3/R region determines the basal level of transcription, whereas sequences within the 5'U3 region enhance transcription four- to fivefold. Deletion mapping for antisense transcription indicates that a 64-base-pair region (nucleotides 47 to 110) within the U3 region is essential for activity. These data indicate that the U3 region contains all the regulatory elements for bidirectional transcription in IAP LTRs.
Mol Cell Biol 1988 Mar
PMID:Functional analysis of the long terminal repeats of intracisternal A-particle genes: sequences within the U3 region determine both the efficiency and direction of promoter activity. 245 71

The enzymes that catalyse the salvage of purines in Entamoeba histolytica trophozoites have been surveyed. Adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4), guanine deaminase (EC 3.5.4.3), adenine phosphoribosyltransferase (PRTase) (EC 2.4.2.7), xanthine PRTase (EC 2.4.2.22) and hypoxanthine PRTase (EC 2.4.2.8) were all detected in cell homogenates but only at low activities, whereas AMP deaminase (EC 3.5.4.6) and guanine PRTase (EC 2.4.2.8) were not found. Phosphorylases (EC 2.4.2.1) active in both anabolic and catabolic directions were present and all nucleosides tested were phosphorylated by kinases (EC 2.7.1.15, EC 2.7.1.20, EC 2.7.1.73). 3'-Nucleotidase (EC 3.1.3.6) and 5'-nucleotidase (EC 3.1.3.5) were found, the former being mainly particulate. Nucleotide interconversion enzymes (adenylosuccinate lyase, EC 4.3.2.2; adenylosuccinate synthetase, EC 6.3.4.4; IMP dehydrogenase, EC 1.2.1.14; GMP synthetase, EC 6.3.5.2 and GMP reductase, EC 1.6.6.8) were not detected. The results suggest that in E. histolytica the main route of nucleotide synthesis is from the individual bases through the actions of phosphorylases and kinases.
Mol Biochem Parasitol 1986 Apr
PMID:Purine-metabolising enzymes in Entamoeba histolytica. 287 91

Guanine is transported into germinated conidia of Neurospora crassa by the general purine base transport system. Guanine uptake is inhibited by adenine and hypoxanthine but not xanthine. Guanine phosphoribosyltransferase (GPRTase) activity was demonstrated in cell extracts of wild-type germinated conidia. The Km for guanine ranged from 29 to 69 micro M in GPRTase assays; the Ki for hypoxanthine was between 50 and 75 micro M. The kinetics of guanine transport differ considerably from the kinetics of GPRTase, strongly suggesting that the rate-limiting step in guanine accumulation in conidia is not that catalyzed by GPRTase. Efflux of guanine or its metabolites appears to have little importance in the regulation of pools of guanine or guanine nucleotides since very small amounts of 14C label were excreted from wild-type conidia preloaded with [8-14C]guanine. In contrast, excretion of purine bases, hypoxanthine, xanthine, and uric acid appears to be a mechanism for regulation of adenine nucleotide pools (Sabina et al., Mol. Gen. Genet. 173:31-38, 1979). No label from exogenous [8-14C]guanine was ever found in any adenine nucleotides, nucleosides, or the base, adenine, upon high-performance liquid chromatography analysis of acid extracts from germinated conidia of wild-type of xdh-l strains. The 14C label from exogenous [8-14C]guanine was found in GMP, GDP, GTP, and the GDP sugars as well as in XMP. Xanthine and uric acid were also labeled in wild-type extracts. Similar results were obtained with xdh-l extracts except that uric acid was not present. The labeled xanthine and XMP strongly suggest the presence of guanase and xanthine phosphoribosyltransferase in germinated conidia.
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PMID:Guanine uptake and metabolism in Neurospora crassa. 617

The nucleotide sequence was determined for the Escherichia coli region containing the gpt gene, which encodes the enzyme xanthine-guanine phosphoribosyl transferase (XGPRT; EC 2.4.2.22). Restriction enzyme and sequence analyses have allowed us to locate precisely the gpt gene (16.9-kDal XGPRT) with respect to other genes in this region, notably phoE. Genes gpt and phoE are pointing towards each other and are separated by about 1840 bp. Available sequence data and protein analyses [Overbeeke et al., J. Mol. Biol. 163 (1983) 513-522, and this paper] indicate the presence, between gpt and phoE, of two additional genes. These genes are oriented the same way as gpt and code for proteins of 49 and 15.7 kDal, respectively. By in vitro transcription with E. coli RNA polymerase and nuclease S1 analysis, we have identified a promoter upstream of gpt. The short intercistronic region between gpt and the 49-kDal protein gene contains a rho-independent termination signal that closely precedes and partially overlaps another promoter. It appears from these data that gpt transcription is essentially monocistronic, giving rise to RNA of approx. 555 nucleotides, whereas the 49-kDal and 15.7-kDal protein genes are transcribed from their own promoter.
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PMID:Structural and functional organization of the gpt gene region of Escherichia coli. 639 1

We have cloned and expressed the full-length gene encoding the hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from the anaerobic protozoan parasite Tritrichomonas foetus. This enzyme is essential in nucleic acid metabolism of T. foetus because the parasite is unable to synthesize purine nucleotides de novo and relies on the HGXPRTase activities for its purine requirements. Initially, a cDNA clone encoding part of the HGXPRTase was isolated by complementation of an Escherichia coli mutant, SO609, with a cDNA library of T. foetus. Northern blot analysis identified a single mRNA band of approximately 700-800 bases. The full-length genomic clone was then isolated and identified to have an open reading frame of 549 bp encoding an 183-amino acid sequence with an estimated size of 21.1 kDa. The sequence is only 27.3% identical to that of the human HGPRTase. The T. foetus HGXPRTase gene was subsequently cloned into the pBAce vector for expression in E. coli. This construct yields completely soluble and enzymatically active recombinant T. foetus HGXPRTase, which constitutes approximately 20% of the total cellular protein of the transformed E. coli. It has the same molecular weight as the authentic native enzyme, and the N-terminal amino acid sequence of the recombinant enzyme is identical to that predicted from the open reading frame. The high expression of this apparently native T. foetus HGXPRTase will provide large quantities of purified protein, necessary for detailed kinetic and structural analysis of this enzyme for its potential value as a target for antitrichomonial chemotherapy. To our knowledge, this is also the first time a gene from T. foetus was cloned and expressed.
Mol Biochem Parasitol 1994 Feb
PMID:Isolation, sequencing and expression of the gene encoding hypoxanthine-guanine-xanthine phosphoribosyltransferase of Tritrichomonas foetus. 800 20

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 phosphoribosyltransferase (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 phosphoribosyltransferase 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 microM, 3.8 microM and 52.4 microM 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 Plasmodium falciparum, which are dimers, with xanthine about 100-times less proficient as a substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Biochem Parasitol 1993 Aug
PMID:The hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus has unique properties. 823 11

The human malaria parasite Plasmodium falciparum is auxotrophic for purines and relies on the purine salvage pathway for the synthesis of its purine nucleotides. Hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) is a key purine salvage enzyme in P. falciparum, making it a potential target for chemotherapy. Previous attempts to purify this enzyme have been unsuccessful because of the difficulty in obtaining cultured parasite material and because of the inherent instability of the enzyme during purification and storage. Other groups have tried to express recombinant P. falciparum HGXPRT but only small amounts of activity were obtained. The successful expression of recombinant P. falciparum HGXPRT in Escherichia coli has now been achieved and the enzyme purified to homogeneity in mg quantities. The measured molecular mass of 26 229+/-2 Da is in excellent agreement with the calculated value of 26232 Da. A method to stabilise the activity and to reactivate inactive samples has been developed. The subunit structure of P. Jilciparum HGXPRT has been determined by ultracentrifugation in the absence (tetramer) and presence (dimer) of KC1. Kinetic constants were determined for 5-phospho-alpha-D-ribosyl-1-pyrophosphate, for the three naturally-occurring 6-oxopurine bases guanine, hypoxanthine, and xanthine and for the base analogue, allopurinol. Differences in specificity between the purified P. falciparum HGXPRT and human hypoxanthine guanine phosphoribosyltransferase enzymes were detected which may be able to be exploited in rational drug design.
Mol Biochem Parasitol 1999 Jan 05
PMID:Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme. 1002 7

We present molecular dynamics (MD) simulations on two enzymes: a human hypoxanthine-guanine-phosphoribosyltransferase (HGPRTase) and its analogue in the protozoan parasite Tritrichomonas foetus. The parasite enzyme has an additional ability to process xanthine as a substrate, making it a hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) [Chin, M. S., and Wang, C. C. (1994) Mol. Biochem. Parasitol. 63 (2), 221-229 (1)]. X-ray crystal structures of both enzymes complexed to guanine monoribosyl phosphate (GMP) have been solved, and show only subtle differences in the two active sites [Eads et al. (1994) Cell 78 (2), 325-334 (2); Somoza et al. (1996) Biochemistry 35 (22), 7032-7040 (3)]. Most of the direct contacts with the base region of the substrate are made by the protein backbone, complicating the identification of residues significantly associated with xanthine recognition. Our calculations suggest that the broader specificity of the parasite enzyme is due to a significantly more flexible base-binding region, and rationalize the effect of two mutations, R155E and D163N, that alter substrate specificity [Munagala, N. R., and Wang, C. C. (1998) Biochemistry 37 (47), 16612-16619 (4)]. In addition, our simulations suggested a double mutant (D106E/D163N) that might rescue the D163N mutant. This double mutant was expressed and assayed, and its catalytic activity was confirmed. Our molecular dynamics trajectories were also used with a structure-based design program, Pictorial Representation Of Free Energy Changes (PROFEC), to suggest parasite-selective derivatives of GMP. Our calculations here successfully rationalize the parasite-selectivity of two novel inhibitors derived from the computer-aided design of Somoza et al. (5) and demonstrate the utility of PROFEC in the design of species-selective inhibitors.
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PMID:Understanding substrate specificity in human and parasite phosphoribosyltransferases through calculation and experiment. 1044 Nov 23

We report the development of a novel dual-modality fusion reporter gene system consisting of Escherichia coli xanthine phosphoribosyltransferase (XPRT) for nuclear imaging with radiolabeled xanthine and Discosoma red fluorescent protein for optical fluorescent imaging applications. The dsRed/XPRT fusion gene was successfully created and stably transduced into RG2 glioma cells, and both reporters were shown to be functional. The level of dsRed fluorescence directly correlated with XPRT enzymatic activity as measured by ribophosphorylation of [14C]-xanthine was in vitro (Ki = 0.124 +/- 0.008 vs. 0.00031 +/- 0.00005 mL/min/g in parental cell line), and [*]-xanthine octanol/water partition coefficient was 0.20 at pH = 7.4 (logP = -0.69), meeting requirements for the blood-brain barrier (BBB) penetrating tracer. In the in vivo experiment, the concentration of [14C]-xanthine in the normal brain varied from 0.20 to 0.16 + 0.05% dose/g under 0.87 + 0.24% dose/g plasma radiotracer concentration. The accumulation in vivo in the transfected flank tumor was to 2.4 +/- 0.3% dose/g, compared to 0.78 +/- 0.02% dose/g and 0.64 +/- 0.05% dose/g in the control flank tumors and intact muscle, respectively. [14C]-Xanthine appeared to be capable of specific accumulation in the transfected infiltrative brain tumor (RG2-dsRed/XPRT), which corresponded to the 585 nm fluorescent signal obtained from the adjacent cryosections. The images of endogenous gene expression with the "sensory system" have to be normalized for the transfection efficiency based on the "beacon system" image data. Such an approach requires two different "reporter genes" and two different "reporter substrates." Therefore, the novel dsRed/XPRT fusion gene can be used as a multimodality reporter system in the biological applications requiring two independent reporter genes, including the cells located behind the BBB.
Mol Imaging 2003 Apr
PMID:Development of a new reporter gene system--dsRed/xanthine phosphoribosyltransferase-xanthine for molecular imaging of processes behind the intact blood-brain barrier. 1296 7


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