Irreversible inhibition of hypoxanthine phosphoribosyltransferase. Further studies on the specificity of periodate-oxidized GMP.

Inactivation of hypoxanthine phosphoribosyltransferase caused by periodate-oxidized GMP is irreversible, even under the conditions of polyacrylamide gel electrophoresis and during affinity chromatography on GMP-Sepharose. Partial binding of the inhibitor to the enzyme protein can be demonstrated on dodecyl sulfate gel electrophoresis: The substrate, phosphoribosyl diphosphate in the presence of Mg2, and the product GMP protect the enzyme against inactivation. Periodate-oxidized GMP, AMP and oxidized purine nucleosides do not influence ribosephosphate pyrophosphokinase, 5'-nucleotidase, purine-nucleoside phosphorylase and guanylate kinase. A variety of other purine nucleosides and nucleotides, tested in their periodateoxidized form, do not lead to a compound comparable or superior to oxidized GMP in its effect on hypoxanthine phosphoribosyltransferase. In an erythrocyte system it is clearly demonstrated that oxidized GMP cannot act across an intact cell membrane.     

Crystallization of the purine salvage enzyme adenine phosphoribosyltransferase

Adenine phosphoribosyltransferase from the protozoan parasite Leishmania donovani has been crystallized in the presence of the substrate Mg²⁺-α-D -5-phosphoribosyl-1-pyrophosphate (PRPP) or the product adenosine-5-monophosphate, as well as in the absence of ligand. These crystals belong to the space group P6₁22 or its enantiomorph P6₅22, with unit cell dimensions of a = b = 64.0 Å, c = 240.5 Å, α = β = 90°, and γ = 120°. The crystals diffract to 1.9 Å. © 1996 Wiley-Liss, Inc.     

The location of purine phosphoribosyltransferase activities in Escherichia coli.

During the preparation of spheroplasts, adenine phosphoribosyltransferase (EC 2.4.2.7) and hypoxanthine phosphoribosyltransferase (EC 2.4.2.8) were released in parallel with cytidine deaminase (EC 3.5.4.5) and uridine phosphorylase (EC 2.4.2.3), which, on other evidence, are considered to be located intracellularly. The two phosphoribosyltransferases and uridine phosphorylase were not significantly associated with purified membrane fractions as was purine nucleoside phosphorylase (EC 2.4.2.1). The effects of the poorly permeable enzyme-inactivating reagents, 4-diazoniumbenzenesulphonate, 7-diazonium-1,3-naphthalene-disulphonate and 2,4,6-trinitrobenzenesulphonate, on Escherichia coli indicate that all the above-mentioned enzymes and also the xanthine-guanine phosphoribosyltransferase [Miller, Ramsey, Krenitsky & Elion (1972) Biochemistry 11, 4723--4731] are located intracellularly.     

Further studies on cytostatic activity of alkoxymethyl purine and pyrimidine acyclonucleosides

The influence of 14 acyclonucleosides, derivatives of adenine, guanine, uracil and thymine on the phosphorylation of dAdo, dGuo, dCyd and dThd occurring in the cytosol of growing amelanotic melanoma transplanted to Syrian hamsters, as well as on inhibition of tumor growth were studied. From among the studied ACNs eight were tested earlier (Modrzejewska et al., 1996, The influence of alkoxymethyl purine and pyrimidine acyclonucleosides on growth inhibition of Kirkman-Robbins hepatoma and possible mechanism of their cytostatic activity, Z. Naturforch. 51c, 75-80); from among the newly synthesized ACNs, 1,3-N,N-diallyloxymethylthymine (AMT2), 1-N-allyloxymethyl-5,6-tetramethyleneuracil (AMUTM), and tested previously 1-N-allyloxymethylthymine (AMT1), administered i.p. in a dose of 0.2 mmol/kg body weight reduce the tumor mass from 0.98 g to 0.64 g +/- 0.11 g (i.e. 35% +/- 12%). 48 hours after i.p. administration of the mentioned ACNs in the same dose a reduction of tumor mass is accompanied by the inhibition of dAMP, dGMP and dTMP synthesis. AMT1 inhibits dThd phosphorylation from 6.2 to 4.22; AMT2 suppresses dAdo, dGuo and dThd phosphorylation by, correspondingly, from 2.8 to 1.7, from 10.8 to 7.5 and from 6.2 to 4.2; AMUTM depresses dAMP synthesis from 2.8 to 1.6 (all data: mumol of 2'dNMP formed per mg of protein per min. x 10(-4)). None of the 14 studied acyclonucleosides influences dCMP synthesis. In vivo, after hydration of allyloxymethyl group to hydroxypropoxymethyl residue (having -CH2OH group), AMT1, AMT2 and AMUTM undergo phosphorylation to corresponding triphosphates. Phosphorylated ACNs are not incorporated into tumor DNA, however they inhibit dAdo, dGuo and dThd incorporation into DNA. It is concluded that ACN triphosphates are not substrates for DNA polymerase but, competing with dATP dGTP and dTTP, inhibit incorporation of these 2'dNTP into DNA and, in consequence, reduce tumor growth, which is presumed to be the main mechanism of cytostatic activity of the studied ACNs.     

Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells

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.     

A conditional mutant deficient in hypoxanthine-guanine phosphoribosyltransferase and xanthine phosphoribosyltransferase validates the purine salvage pathway of Leishmania donovani

Leishmania donovani cannot synthesize purines de novo and express a multiplicity of enzymes that enable them to salvage purines from their hosts. Previous efforts to generate an L. donovani strain deficient in both hypoxanthine-guanine phosphoribosyl-transferase (HGPRT) and xanthine phosphoribosyltransferase (XPRT) using gene replacement approaches were not successful, lending indirect support to the hypothesis that either HGPRT or XPRT is crucial for purine salvage by the parasite. We now report the genetic confirmation of this hypothesis through the construction of a conditional delta hgprt/delta xprt mutant strain that exhibits an absolute requirement for 2'-deoxycoformycin, an inhibitor of the leishmanial adenine aminohydrolase enzyme, and either adenine or adenosine as a source of purine. Unlike wild type parasites, the delta hgprt/delta xprt strain cannot proliferate indefinitely without 2'-deoxycoformycin or with hypoxanthine, guanine, xanthine, guanosine, inosine, or xanthosine as the sole purine nutrient. The delta hgprt/delta xprt mutant infects murine bone marrow-derived macrophages <5% as effectively as wild type parasites and cannot sustain an infection. These data establish genetically that either HGPRT or XPRT is absolutely essential for purine acquisition, parasite viability, and parasite infectivity of mouse macrophages, that all exogenous purines are funneled to hypoxanthine and/or xanthine by L. donovani, and that the purine sources within the macrophage to which the parasites have access are HGPRT or XPRT substrates.     

Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase. Stability studies on the product-activated enzyme

Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.     

Further studies on the mechanism of phenol-sulfuric acid reaction with furaldehyde derivatives

Even though the chromogens formed from mannose and galactose showed comparable absorbances at 480 nm in the conventional (developer present during heat of dilution) and modified (developer reacted at room temperature after cooling; epsilon mannose = 13,700, galactose = 14,000) phenol-sulfuric acid reactions, shoulders in the region 420-430 nm were prominent in the former method. Fucose was 10 times less reactive in the modified method (epsilon = 800) than in the conventional method. 2-Formyl-5-furan sulfonic acid reacted equally efficiently in the two methods (epsilon = 40,800). 5-Methyl-2-furaldehyde, unlike the sulfonate derivative or 5-hydroxymethyl-2-furaldehyde, required heat for condensation with phenol. 2-Furaldehyde dimethylhydrazone reacted 25 times better to form a chromogen (epsilon = 40,500) in the modified phenol-sulfuric acid method. The possible roles of intermediates between hexoses and furaldehydes in forming chromogens and the effect of substitution at the 2- and 5-positions of furaldehyde on the rates of condensation with phenol for the observed differences between the conventional and the modified methods are discussed.     

Chlorination studies. IV. The reaction of aqueous hypochlorous acid with pyrimidine and purine bases

The labile intermediates and stable end products formed by the reaction of aqueous HOCl with thymine, uracil, 5-Br-uracil, N,N-dimethyluracil and 6-methyluracil have been identified. The purine ring system of guanine, adenine and xanthine was more resistant to attack by aqueous HOCl and reaction times of one week resulted in the formation of parabanic acid. Caffeine and theophylline under similar reaction conditions yielded N,N-dimethylparabanic acid.     

Further studies on the thymocyte stimulating factor.

The thymocyte-stimulating activity produced by spleen cells cultured in the presence of PHA (cell. Immunol.17, 495, 1975) was further investigated. It was found that this activity is caused by a factor having the chemical properties of a protein with a molecular weight of 30,000–32,000 that, at high concentration, probably dimerizes to a molecule of molecular weight about 55,000 as measured by Sephadex gel filtration. This factor is produced also in mixed lymphocyte cultures of CBA and C57 spleen cells. The observation that the factor produced in the presence of PHA and the factor produced in the mixed lymphocyte cultures have the same molecular weight and the same rate of denaturation at two different temperatures suggests that they are the same, or very similar, substance. The fact that spleen cells of nu/nu mice do not produce thymocyte-stimulating factor suggests that thymus-dependent cells are involved in the production of this factor. This assumption is consistent with the observation that spleen cells of mice less than two weeks old (which show a very small response to PHA) do not produce significant amounts of thymocyte-stimulating factor. On the other hand, results of experiments with hydrocortisone-injected mice suggest that the target cell for thymocyte-stimulating factor is the immature, cortisone-sensitive thymocyte. The properties of thymocyte-stimulating factor are discussed and compared to those of factors with similar actions reported in the literature.     

Further studies on the genus Berkeleya Grev.

Detailed light and electron microscopical studies on the diatom genus Berkeleya Grev. clarify relationships between the four species and support the separtion of B. fragilis and B. micans as two distinct taxa.