Toxoplasma gondii tachyzoites possess an unusual plasma membrane adenosine transporter

Nucleoside transport may play a critical role in successful intracellular parasitism by Toxoplasma gondii. This protozoan is incapable of de novo purine synthesis, and must salvage purines from the host cell. We characterized purine transport by extracellular T. gondii tachyzoites, focusing on adenosine, the preferred salvage substrate. Although wild-type RH tachyzoites concentrated [³H]adenosine 1.8-fold within 30 s, approx. half of the [³H]adenosine was converted to nucleotide, consistent with the known high parasite adenosine kinase activity. Studies using an adenosine kinase deficient mutant confirmed that adenosine transport was non-concentrative. [¹⁴C]Inosine, [¹⁴C]hypoxanthine and [³H]adenine transport was also rapid and non-concentrative. Adenosine transport was inhibited by dipyridamole (IC₅₀ approx. 0.7 μM), but not nitrobenzylthioinosine (15 μM). Transport of inosine, hypoxanthine and adenine was minimally inhibited by 10 μM dipyridamole, however. Competition experiments using unlabeled nucleosides and bases demonstrated distinct inhibitor profiles for [³H]adenosine and [¹⁴C]inosine transport. These results are most consistent with a single, dipyridamole-sensitive, adenosine transporter located in the T. gondii plasma membrane. Additional permeation pathways for inosine, hypoxanthine, adenine and other purimes may also be present.     

Purification and characterization of a soluble nucleoside diphosphate kinase in Trypanosoma cruzi

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The K_m values of the enzyme for ATP, GDP and dTDP were 0.2 ± 0.008 mM, 0.125 ± 0.012 mM and 0.4 ± 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65°C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [γ-³²P]ATP or thiophosphorylated with [³⁵S]GTPγS. The incubation of the ³²P-labelled phosphoenzyme with unlabelled nucleoside 5′-diphosphates resulted in the formation of ³²P-labelled nucleoside 5′-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5′-diphosphates, GTP was preferentially formed.     

Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasite's host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.     

Purification of hypoxanthine-guanine phosphoribosyltransferase of Plasmodium lophurae

Hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) was isolated from the malarial parasite, Plasmodium lophurae. The apparent pI, as determined by chromatofocusing, was 7.6. The native molecular weight was 79,000. The pH profile of HGPRT exhibited a broad pH optimum. With hypoxanthine as substrate maximal activity was achieved from pH 6.0-10.0, and with guanine as substrate maximal activity occurred from pH 7.5-9.5. The enzyme exhibited Michaelis-Menten kinetics with all substrates. The Km values were 3.8 microM (hypoxanthine), 2.4 microM (guanine), 6.2 microM (6-mercaptopurine), 7.6 microM (6-thioguanine), and 360 microM (8-azahypoxanthine). 6-Thioinosine, 9-beta-arabinofuranosylhypoxanthine, 6-chloropurine, xanthine and azaguanine were inhibitors of the P. lophurae enzyme. From the substrate and inhibitor data it appears that the sixth position on the purine ring plays a major role in enzyme activity.     

Inhibitory action of the antitumor agent lonidamine on mitochondrial respiration of Trypanosoma cruzi and T. brucei

The antitumor and antispermatogenic agent lonidamine inhibits Trypanosoma cruzi epimastigotes growth in culture with an ID₅₀ around 80 μM. The main site of action appears to be the mitochondria, where the rate of uncoupled respiration was inhibited in 50% at a similar lonidamine concentration (50 μM). Hexokinase (the other point where lonidamine inhibits tumor energy metabolism) was not sensitive to this drug. Lonidamine also inhibited uncoupled respiration in T. brucei procyclic trypomastigotes, suggesting a common mechanism of action with T. cruzi. When lonidamine was added to T. brucei trypomastigotes, there was little effect on the CN-insensitive respiration, demonstrating that at least in T. brucei glycolysis is not affected by the drug.     

SfiI and NotI polymorphisms in Theileria stocks detected by pulsed field gel electrophoresis

DNAs of Theileria parva parva, T. p. lawrencei, T. p. bovis and Theileria mutans stocks, from Kenya, Uganda, Zanzibar and Zimbabwe were digested with either SfiI or NotI and analysed using contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE). The SfiI-digested T. parva genomic DNA resolved into approximately 30 fragments while the NotI digestion produced between 4–7 bands. The summation of the sizes of SfiI fragments gave an estimate of 9–10×10⁶ base pairs for the size of the T. parva genome. Heterogeneity within T. p. parva Muguga, Pemba/Mnarani and Mariakani stocks was detected. All the T. parva stocks analysed showed SfiI and NotI restriction fragment length polymorphisms (RFLP). Hybridisation of 5 SfiI-digested T. parva DNAs with a Plasmodium berghei telomeric repeat probe suggested that most of the polymorphisms and heterogeneity occurred in the telomeric or sub-telomeric regions of the genome. The recognition by the Plasmodium telomeric probe of 7–8 strongly hybridising SfiI bands indicates that the T. parva genome may possess at least 4 chromosomes. The T. mutans genome was cut frequently with the above enzymes resulting in large numbers of fragments predominantly below 50 kb, thus suggesting either a much higher G+C content than T. parva or the presence of highly reiterated G+C-rich regions.     

Affinity of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: I. In vitro adsorption studies

In previous papers, we described insoluble polystyrene derivatives which exhibit a heparin-like antithrombic activity in plasma. In order to ascertain the heparin-like mechanism of this activity we have studied the interactions of thrombin and antithrombin III with two polymers of this series: sulphonated polystyrene and sulphonate-glutamic acid sulphonamide polystyrene. The adsorption was measured using purified enzyme and enzyme inhibitor and polymer beads whose average diameter was about 25 μm. The maxima of adsorption approximately correspond to a monolayer of protein. The results are discussed with respect to the most common isotherms used in chemisorption and the affinities of the enzyme and its inhibitor for both materials are evaluated: k_T- 10⁷(M/I)⁻¹, k_AT- 3.10⁵(M/I)⁻¹.     

Purification and properties of monomeric (G1) forms of acetylcholinesterase secreted by Nippostrongylus brasiliensis

Acetylcholinesterase (AChE) activity secreted by Nippostrongylus brasiliensis was resolved by sucrose density centrifugation and gel permeation chromatography in single peaks estimated at 4.3 S and 60–85 kDa, respectively. Sedimentation was unaffected by the inclusion of detergent. AChE was purified by affinity chromatography on 9-[N^β-(-aminocaproyl)-β-aminopropylamino]-acridinium bromide hydrobromide-coupled sepharose 4B. Three forms of the enzyme (A, B and C) were distinguished by non-denaturating polyacrylamide gel electrophoresis, and displayed apparent masses of 74, 69 and 71 kDa respectively when resolved by SDS-PAGE. All three isoforms showed a preference for acetylthiocholine (ASCh) as substrate. They were highly sensitive to inhibition by the AChE-specific inhibitor bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with inhibitor concentration reducing initial activity by 50% (IC₅₀) between 0.1 and 0.8 μM, but activity was unaffected by tetramonoisopropylpyrophosphortetramide (iso-OMPA) at concentrations up to 10 mM. We conclude that the secreted enzymes are authentic AChEs of hydrophilic monomeric (G₁) form and broadly similar properties, but which can be distinguished by molecular mass, inhibitor sensitivities and the degree of excess substrate inhibition.     

Purine salvage by Tritrichomonas foetus

The anaerobic protozoon Tritrichomonas foetus was found incapable of de novo purine synthesis by its failure to incorporate radiolabeled glycine or formate into the nucleotide pool. It had, on the other hand, high activities in incorporating adenine, hypoxanthine or inosine. Radiolabel pulse-chase experiments indicated that adenine, hypoxanthine and inosine all entered the pool through conversion to IMP. The parasite contained hypoxanthine phosphoribosyl transferase, adenine deaminase and inosine phosphorylase, but no adenine phosphoribosyl transferase, inosine kinase or inosine phosphotransferase activity. Adenine and inosine had to be converted to hypoxanthine before incorporation. Adenosine was also rapidly converted to hypoxanthine in T. foetus cell-free extracts, but the presence of adenosine kinase in the parasite allowed some conversion of adenosine directly to AMP. Guanine and xanthine were directly incorporated into GMP and XMP, probably due to the guanine and xanthine phosphoribosyl transferase. There were also strong enzyme activities which convert guanosine to guanine and guanine to xanthine. A guanosine phosphotransferase was found in the 10(5) X g sedimentable fraction of T. foetus, and was capable of converting some guanosine to GMP. This network of T. foetus purine salvage suggests the importance of hypoxanthine-guanine-xanthine phosphoribosyl transferase activities in the parasite.     

Acquisition of lipoproteins in the procyclic form of Trypanosoma brucei

The procyclic form of Trypanosoma brucei binds and internalizes bovine high density lipoprotein (HDL) particles in a saturable process; the binding and uptake of ¹²⁵I-labeled HDL are inhibited by excess unlabeled HDL. We calculated that each procyclic trypanosome exposes ≈1.0×10⁶ binding sites for bovine HDL, with an equilibrium dissociation constant (K_d) of ≈1.26×10⁻⁷ M. Uptake of HDL particles does not occur at 4°C. At 28°C, a significant amount of the internalized HDL particles were efficiently degraded through a process that is sensitive to the presence of 50 μM chloroquine. These results suggested that the uptake of HDL particles in procyclic T. brucei may occur via receptor mediated endocytosis, leading to proteolytic degradation of the particles in an acidic and endocytic compartment.     

Purification and characterization of Plasmodium falciparum hypoxanthine-guanine-xanthine phosphoribosyltransferase and comparison with the human enzyme

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.     

Adenosine A1 receptors in human hippocampus: inhibition of [H]8-cyclopentyl-1,3-dipropylxanthine binding by antagonist drugs

Adenosine A₁ receptors were visualized in human hippocampus using [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as a radioactive ligand probe. The receptor antagonists caffeine, the xanthine derivative KFM 19 and the carbamazepine analogue oxcarbazepine displaced [³H]DPCPX binding homogeneously without any marked difference between the individual layers in the investigated hippocampal subregions (n = 4). K_i's in the individual layers were in a range between 8.5 ± 6.5 μM and 18.9 ± 16.0 μM for caffeine and 11.5 ± 2.8 nM and 18.1 ± 14.1 nM for KFM 19.K_i's could not be calculated for oxcarbazepine as the IC₅₀'s were greater than 100 μM with estimated IC₂₅'s varying between 51.2 ± 53.3 μM and 179.9 ± 89.9 μM. Antagonism of endogenous adenosine at A₁ receptors may thus explain part of the clinical effects of caffeine in humans and possibly exclusively the behavioral effects of KFM 19 in non-human primates.     

The trypanocidal Cape buffalo serum protein is xanthine oxidase.

Plasma and serum from Cape buffalo (Syncerus caffer) kill bloodstream stages of all species of African trypanosomes in vitro. The trypanocidal serum component was isolated by sequential chromatography on hydroxylapatite, protein A-G, Mono Q, and Superose 12. The purified trypanocidal protein had a molecular mass of 150 kDa, and activity correlated with the presence of a 146-kDa polypeptide detected upon reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid sequences of three peptide fragments of the 146-kDa reduced polypeptide, ligand affinity and immunoaffinity chromatography of the native protein, and sensitivity to pharmacological inhibitors, identified the trypanocidal material as xanthine oxidase (EC 1.1.3.22). Trypanocidal activity resulted in the inhibition of trypanosome glycolysis and was due to H2O2 produced during catabolism of extracellular xanthine and hypoxanthine by the purine catabolic enzyme.     

Purification and characterization of the extracellular acid phosphatase of Leishmania donovani

A tartrate sensitive acid phosphatase activity was purified from culture supernatants of Leishmania donovani promastigotes grown in a macromolecule-free defined medium. Purification was accomplished by ultrafiltration, lentil lectin affinity chromatography, and sucrose density gradient ultracentrifugation. This enzyme was determined to be an acid glycoprotein containing 0.37 mg hexose per mg protein. A molecular weight of 134 000 by sucrose density gradient centrifugation was observed, although on molecular sieve chromatography the enzyme eluted with an apparent molecular weight of>700 000. The specific activity of the purified enzyme was>200 μmol min⁻¹(mg protein)⁻¹ when assayed with 4-methylumbelliferylphosphate as the substrate. In addition to various hexose phosphates, the enzyme hydrolyzed phosphorylated amino acids, in particular phosphotyrosine. The purified enzyme was heterodisperse with respect to both protein and activity staining patterns upon polyacrylamide gel electrophoresis.     

N,N′-Thiophene-substituted polyamine analogs inhibit mammalian host cell invasion and intracellular multiplication of Trypahosoma cruzi

We studied the effects of two, N,N′-thiophene-substituted polyamine analogs (MDL 28302 and MDL 29431) on the capacities of Trypanosoma cruzi, the etiologic agent of Chagas' disease, to invade and multiply within a mammalian host cell. Both compounds inhibited infectivity significantly in a time- and concentration-dependent manner. This inhibition resulted from a selective effect on the parasite, because pretreatment of T. cruzi but not host cell cultures with either MDL 28302 or MDL 29431 reduced infectivity. The parasite gradually recovered its infective capacity after removal of unincorporated polyamine analog, denoting the reversible nature of the inhibitory effect. Some biochemical modification of MDL 28302 and MDL 29431 appeared to be required for their inhibitory activities to be exerted, since the effects of these drugs on T. cruzi infectivity were abrogated by MDL 72527, a drug known to inhibit polyamine oxidase (PAO) activity specifically. Supporting the notion of that products of MDL 28302 and MDL 29431 oxidation by PAO were involved in the activity of these compounds was the finding that PAO competitive substrates (N¹Lacetylspermine and N¹-acetylspermidine) also abolished the inhibition of T. cruzi infectivity mediated by MDL 28302 or MDL 29431. However, we can not rule out that MDL 72527 and the PAO competitive substrates might have altered an alternative mechanism because no significant polyamine oxidase activity could be demonstrated in preparations of lysed or intact T. cruzi in assays monitoring conversion of [¹⁴C]spermine to [¹⁴C]spermidine. When either MDL 28302 or MDL 29431 was added to infected cell cultures, a marked reduction in the rate of intracellular parasite growth ensued. The significance of the finding that N,N′-thiophene-substituted polyamine analogs inhibit cell invasion and cytoplasmic replication by T. cruzi resides in the fact that this pathogenic parasite requires a cytoplasmic localization to replicate in mammalian hosts.     

Purine metabolism in Trypanosoma cruzi

Culture forms of Trypanosoma cruzi are incapable of synthesizing purines de novo from formate, glycine, or serine and require an exogenous purine for growth. Adenine, hypoxanthine, guanine, xanthine and their respective ribonucleosides are equal in their abilities to support growth. Radiolabeled purine bases, with the exception of guanine, are stable and are converted to their respective ribonucleotides directly by phosphoribosyltransferase activity. Guanine is both converted to its ribonucleotide and deaminated to xanthine. Purine nucleosides are not hydrolysed to any extent but are converted to their respective ribonucleotides. This conversion may involve a rate-limiting ribonucleoside cleaving activity or a purine nucleoside kinase or phosphotransferase activity. The apparent order of salvage efficiency for the bases and their respective ribonucleosides is adenine > hypoxanthine > guanine > xanthine.     

Heparin prevents formation of the human C3 amplification convertase by inhibiting the binding site for B on C3b

Fluid-phase heparin prevents generation of the C3 amplification convertase of human complement, C3b, Bb most likely by inhibiting the formation of the bimolecular complex between cell-bound C3b and B. The effects of heparin on the binding of B to C3b was examined using ¹²⁵I-labelled B and C3b-bearing sheep erythrocytes (E^sC3b). In the absence of heparin, B found to E^sC3b with an affinity of 0.5–1 × 10⁶ M⁻¹ in the presence of 5 mM Mg²⁺. Incremental amounts of heparin (100–700 μg/107 E^sC3b) inhibited the binding of ¹²⁵I-B to C3b in a dose-dependent manner. Scatchard analysis of the binding data in the presence of four inhibitory concns of heparin revealed that heparin did nor affect the binding affinity of B for C3b but decreased the number of C3b sites recognized by B on the cells. No inhibition of binding occurred in the presence of totally (N- and O-) desulfated heparin which has no anticomplementary activity. These results demonstrate that heparin prevents generation of the C3 amplification convertase by binding to cell-bound C3b and masking the binding site for B on C3b.     

Purine metabolism in the intact sporozoites and merozoites of Eimeria tenella

Intact Eimeria tenella sporozoites and merozoites did not incorporate radiolabeled formate or glycine into their purine nucleotides suggesting a lack of de novo purine synthesis. However, [U-14C]glucose was incorporated into the cellular purine and pyrimidine nucleotide pools of both forms probably via conversion to radiolabeled ribose-1-phosphate and/or 5'-phosphoribosyl-1-alpha-pyrophosphate and the resulting action of various purine and pyrimidine salvage enzymes. Both forms of the parasite salvaged radiolabeled purine bases and nucleosides in a similar fashion. These purines were incorporated into ribonucleotides and into RNA and DNA. Adenine and inosine were transformed to hypoxanthine. Adenosine was converted to both inosine and hypoxanthine. Hypoxanthine and xanthine were not oxidized to uric acid but were metabolized to nucleotides. Guanosine was cleaved to guanine; guanine was deaminated to xanthine. The results demonstrate the presence of several purine salvage pathways. Purine phosphoribosylating and nucleoside phosphorylating activities as well as purine nucleoside cleaving and adenosine, adenine and guanine deaminating activities were evident. The metabolic evidence suggests the enzymes required to convert the newly formed nucleoside monophosphates to ATP and GTP were present also.     

Genetic evidence for the essential role of PfNT1 in the transport and utilization of xanthine, guanine, guanosine and adenine by Plasmodium falciparum

The malaria parasite, Plasmodium falciparum, is unable to synthesize the purine ring de novo and is therefore wholly dependent upon purine salvage from the host for survival. Previous studies have indicated that a P. falciparum strain in which the purine transporter PfNT1 had been disrupted was unable to grow on physiological concentrations of adenosine, inosine and hypoxanthine. We have now used an episomally complemented pfnt1Delta knockout parasite strain to confirm genetically the functional role of PfNT1 in P. falciparum purine uptake and utilization. Episomal complementation by PfNT1 restored the ability of pfnt1Delta parasites to transport and utilize adenosine, inosine and hypoxanthine as purine sources. The ability of wild-type and pfnt1Delta knockout parasites to transport and utilize the other physiologically relevant purines adenine, guanine, guanosine and xanthine was also examined. Unlike wild-type and complemented P. falciparum parasites, pfnt1Delta parasites could not proliferate on guanine, guanosine or xanthine as purine sources, and no significant transport of these substrates could be detected in isolated parasites. Interestingly, whereas isolated pfnt1Delta parasites were still capable of adenine transport, these parasites grew only when adenine was provided at high, non-physiological concentrations. Taken together these results demonstrate that, in addition to hypoxanthine, inosine and adenosine, PfNT1 is essential for the transport and utilization of xanthine, guanine and guanosine.