Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.4.2.7 (adenine phosphoribosyltransferase)
692 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activities from promastigotes of Leishmania donovani have been purified to homogeneity using ammonium sulfate precipitation, DEAE-cellulose exclusion, and either AMP-agarose (APRTase) or GTP-agarose (HGPRTase) affinity chromatography. The specific activities of the affinity-purified APRTase and HGPRTase fractions were 326-fold and 1341-fold greater than those in the 40-80% ammonium sulfate precipitate, respectively. The purified APRTase migrated as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels with a size of 29 kDa, while HGPRTase was also determined to be homogeneous by SDS gel electrophoresis with a size of 24 kDa. In addition, a mutant cell line, APPB2, partially deficient in APRTase activity, still contained quantities of purifiable APRTase protein, while a clonal secondary derivative of the APPB2 cell line that is completely deficient in APRTase activity, APPB2-640A3, failed to express purifiable APRTase protein. The homogeneous enzymes possessed apparent Km values for their nucleobase substrates between 2.0 and 5.0 microM, and both enzymes were inhibited by their immediate or ultimate reaction endproducts, APRTase by AMP and PPi and HGPRTase by GMP, GTP, and PPi. The generation of homogeneous preparations of APRTase and HGPRTase protein will serve as a prerequisite for the generation of immunological and molecular biological probes to analyze the leishmanial phosphoribosyltransferases.
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PMID:Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase activities from Leishmania donovani. 270 89

Giardia lamblia, a flagellated parasitic protozoan and the causative agent of giardiasis, lacks de novo purine biosynthesis and exists on salvage of adenine and guanine by adenine phosphoribosyltransferase and guanine phosphoribosyltransferase. Guanine phosphoribosyltransferase from G. lamblia crude extracts has been purified to apparent homogeneity by Sephacryl S-200 gel filtration followed by C-8-GMP-agarose and 2',3'-GMP-agarose affinity chromatography, resulting in an overall recovery of 77% and a purification of 83,000-fold. The molecular weight of the native enzyme as estimated by gel filtration and isokinetic sucrose gradients was found to be 58,000-63,000, with a subunit molecular weight of approximately 29,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mono P chromatofocusing chromatography gives rise to a major activity peak eluting from the column at a pH of 6.75 and two minor activity peaks at pH of 5.3 and 5.2. Hypoxanthine and xanthine can be recognized by the enzyme as substrates but at Km values 20 times higher than that observed with guanine. G. lamblia guanine phosphoribosyltransferase is immunologically distinct from human hypoxanthine-guanine phosphoribosyltransferase and Escherichia coli xanthine-guanine phosphoribosyltransferase, and G. lamblia DNA fragments are incapable of hybridizing with mouse neuroblastoma hypoxanthine-guanine phosphoribosyltransferase DNA or E. coli xanthine phosphoribosyltransferase DNA under relatively relaxed conditions. All evidence presented suggests that G. lamblia guanine phosphoribosyltransferase may be qualified as a potential target for antigiardiasis chemotherapy.
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PMID:Purification and characterization of guanine phosphoribosyltransferase from Giardia lamblia. 308 75

Of 142 purines, purine nucleosides, and analogues tested for inhibition of growth of Escherichia coli B Hill, 45 were active. Of these, 27 were evaluated for inhibition of other E. coli lines, including those resistant to 6-thioguanine, 2-fluoroadenosine, 2,6-diaminopurine, or 6-mercaptopurine. Most toxic to the parent lines were 2-fluoroadenosine, 2-fluoroadenine, 2-fluoro-5'-deoxyadenosine, adenosine, 6-thioguanosine, 6-thioguanine, 6-mercaptopurine, 6-mercaptopurine ribonucleoside, 2-azaadenine, 2'-deoxyinosine, 6-N-aminoadenine, and inosine. Hypoxanthine was strongly inhibitory only to E. coli B Hill. Evidence regarding the substrate specificity of the three purine phosphoribosyltransferases was obtained by assaying for these enzymes in extracts of the various cell lines and by cross-resistance studies. The line selected for resistance to 6-thioguanine had low guanine phosphoribosyltransferase activity (guanosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) and was deficient in activity for xanthine and 6-thioguanine. The lines selected for resistance to 2-fluoroadenosine and 2,6-diaminopurine were deficient in adenine phosphoribosyltransferase activity (adenosine monophosphate: pyrophosphate phosphoribosyltransferase, EC 2.4.2.7), and that selected for resistance to 6-mercaptopurine had low hypoxanthine phosphoribosyltransferase activity and undetectable activity with 6-mercaptopurine as a substrate. Purine, 6-methylpurine, 2-fluoroadenine, 2,6-diaminopurine, and 2-azaadenine were classified as adenine analogues; 6-mercaptopurine and 8-aza-2,6-diaminopurine, as hypoxanthine analogues; and 6-thioguanine and 2-amino-6-chloropurine, as analogues of guanine. The inhibition of bacterial growth by hypoxanthine, inosine, 2'-deoxyinosine, or adenosine was prevented by small amounts of thiamine or by relatively high concentrations of either cytidine or uridine. Cytidine also reversed the inhibition by some purine and purine ribonucleoside analogues. Orotate phosphoribosyltransferase (OMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.10), a possible site of action for these compounds, was not inhibited directly by the toxic agents.
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PMID:Use of Escherichia coli mutants to evaluate purines, purine nucleosides, and analogues. 459 16

1. The purine bases adenine, hypoxanthine and guanine were rapidly incorporated into the nucleotide fraction of Ehrlich ascites-tumour cells in vivo. 2. The reaction of 5'-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase from ascites-tumour cells (K(m) 6.5-11.9mum) was competitively inhibited by AMP, ADP, ATP and GMP (K(i) 7.5, 21.9, 395 and 118mum respectively). Similarly the reactions of 5'-phosphoribosyl pyrophosphate with both hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase (K(m) 18.4-31 and 37.6-44.2mum respectively) were competitively inhibited by IMP (K(i) 52 and 63.5mum) and by GMP (K(i) 36.5 and 5.9mum). 3. The nucleotides tested as inhibitors did not appreciably compete with the purine bases in the phosphoribosyltransferase reactions. 4. It was postulated that the purine phosphoribosyltransferases of Ehrlich ascites-tumour cells may be effectively separated from the adenine nucleotide pool of these cells.
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PMID:Inhibition of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells by purine nucleotides. 596 81

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.
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PMID:Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells. 606 4

1. Adenine phosphoribosyltransferase was protected from inactivation on heating at 55 degrees by the presence of 5-phosphoribosyl pyrophosphate. ATP, adenine, AMP or GMP had no protective effect on the activity of this enzyme. The presence of either 5-phosphoribosyl pyrophosphate or ATP did not protect adenine phosphoribosyltransferase against the loss of ATP stimulation obtained by heating at 55 degrees . 2. At pH5.3 and 6.0 adenine phosphoribosyltransferase was stimulated by a narrow range of ATP concentration (15-25mum). At pH6.5 and 7.0 maximum stimulation was obtained with 25-30mum-ATP, and at pH7.4, 8.2 and 8.85 maximum stimulation was obtained over a wide range of ATP concentrations (60-200mum). With extracts that had been heated for 30min. at 55 degrees no stimulation was observed at either pH5.3 or 7.4 with ATP concentrations up to 100mum. 3. Short periods of heating at 55 degrees (1, 2 or 5min.) increased the stimulation of adenine phosphoribosyltransferase obtained with various concentrations of ATP. 4. The addition of CTP, GTP, deoxy-GTP, deoxy-TTP or XTP to assay mixtures resulted in weak stimulation of adenine-phosphoribosyltransferase activity. 5. It is suggested that there are at least three different forms of adenine phosphoribosyltransferase, each with a different affinity for ATP.
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PMID:Stimulation of adenine phosphoribosyltransferase by adenosine triphosphate and other nucleoside triphosphates. 606 33

A family is described in which four affected males, spanning two generations, have hyperuricemia and gout accompanied by hematuria but are without severe neurologic involvement. The affected males were found to have markedly reduced levels of erythrocytic hypoxanthine-guanine phosphoribosyltransferase (HGPRT) activity; these were 5-12% with hypoxanthine and 0.5-3% with guanine as compared to controls. Erythrocytic adenine phosphoribosyltransferase (APRT) was approximately three-fold elevated in the affected individuals. The residual HGPRT activity in affected males enabled characterization of some of the properties of this mutation. The apparent Michaelis constants (km) for both hypoxanthine and guanine were essentially unchanged, whereas the km for PP-ribose-P was approximately 10-20-fold elevated for all four affected males. The enzyme was more sensitive to product inhibition by IMP and GMP than controls, and exhibited greater thermal lability at 65 degrees C than found with control lysates.
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PMID:Partial deficiency of hypoxanthine-guanine phosphoribosyltransferase with reduced affinity for PP-ribose-P in four related males with gout. 620 22

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been proven to be a target for potential anti-tritrichomonial chemotherapy. Using a structure-based approach, the base-binding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determining the roles of different active-site residues in purine specificity would form the basis for designing specific inhibitors toward the parasitic enzyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longer recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recognize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the Kms for GMP and XMP (but not IMP). This change leads to increased kcats for the forward reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr156, resulted from mutations of Arg155, bring little effect on the hydrophobic interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which involve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain carboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 was proven to be the primary determinant in conferring the specificity of the enzyme toward 6-oxopurines. By substituting the lysine residue for a serine, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xanthine as its substrates. Adenine and hypoxanthine are equivalent substrates for the mutant enzyme with similar Kms of 34.6 and 38.0 microM, respectively. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AMP with an estimated Kis of 25.4 microM against alpha-D-5-phosphoribosyl-pyrophosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetusHGXPRTase into either a HGPRTase or a genuine AHGXPRTase.
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PMID:Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein. 984 28

1. A strain of Ehrlich ascites-tumour cells that showed little inhibition of growth in the presence of 6-mercaptopurine accumulated less than 5% as much 6-thioinosine 5'-phosphate in vivo, in the presence of 6-mercaptopurine, as did the sensitive strain from which it was derived. 2. Specific activities of the phosphoribosyltransferases that convert adenine, guanine, hypoxanthine and 6-mercaptopurine into AMP, GMP, IMP and 6-thioinosine 5'-phosphate were similar in extracts of the resistant and the sensitive cells. 3. As found previously with sensitive cells, 6-mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase and hypoxanthine phosphoribosyltransferase from the resistant cells and does not inhibit the adenine phosphoribosyltransferase from these cells. Michaelis constants and inhibitor constants of the purine phosphoribosyltransferases from resistant cells did not differ significantly from those measured with the corresponding enzymes from sensitive cells. 4. Resistance to 6-mercaptopurine in this case is probably not due to qualitative or quantitative changes in these transferases.
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PMID:INHIBITION BY 6-MERCAPTOPURINE OF PURINE PHOSPHORIBOSYLTRANSFERASES FROM EHRLICH ASCITES-TUMOUR CELLS THAT ARE RESISTANT TO THE DRUG. 1434 51

To find general metabolic profiles of purine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, we looked at the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers. The activities of key enzymes in potato tuber extracts were also studied. Of the precursors for the intermediates in de novo purine biosynthesis, [(14)C]formate, [2-(14)C]glycine and [2-(14)C]5-aminoimidazole-4-carboxyamide ribonucleoside were metabolised to purine nucleotides and were incorporated into nucleic acids. The rates of uptake of purine ribo- and deoxyribonucleosides by the disks were in the following order: deoxyadenosine > adenosine > adenine > guanine > guanosine > deoxyguanosine > inosine > hypoxanthine > xanthine > xanthosine. The purine ribonucleosides, adenosine and guanosine, were salvaged exclusively to nucleotides, by adenosine kinase (EC 2.7.1.20) and inosine/guanosine kinase (EC 2.7.1.73) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Inosine was also salvaged by inosine/guanosine kinase, but to a lesser extent. In contrast, no xanthosine was salvaged. Deoxyadenosine and deoxyguanosine, was efficiently salvaged by deoxyadenosine kinase (EC 2.7.1.76) and deoxyguanosine kinase (EC 2.7.1.113) and/or non-specific nucleoside phosphotransferase (EC 2.7.1.77). Of the purine bases, adenine, guanine and hypoxanthine but not xanthine were salvaged for nucleotide synthesis. Since purine nucleoside phosphorylase (EC 2.4.2.1) activity was not detected, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine/guanine phosphoribosyltransferase (EC 2.4.2.8) seem to play the major role in salvage of adenine, guanine and hypoxanthine. Xanthine was catabolised by the oxidative purine degradation pathway via allantoin. Activity of the purine-metabolising enzymes observed in other organisms, such as purine nucleoside phosphorylase (EC 2.4.2.1), xanthine phosphoribosyltransferase (EC 2.4.2.22), adenine deaminase (EC 3.5.4.2), adenosine deaminase (EC 3.5.4.4) and guanine deaminase (EC 3.5.4.3), were not detected in potato tuber extracts. These results suggest that the major catabolic pathways of adenine and guanine nucleotides are AMP --> IMP --> inosine --> hypoxanthine --> xanthine and GMP --> guanosine --> xanthosine --> xanthine pathways, respectively. Catabolites before xanthosine and xanthine can be utilised in salvage pathways for nucleotide biosynthesis.
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PMID:Profiles of purine biosynthesis, salvage and degradation in disks of potato (Solanum tuberosum L.) tubers. 1684 29


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