scholarly journals Identification, partial purification and inhibition by guanine analogues of a novel enzymic activity which phosphorylates guanosine to GMP in the protozoan parasite Eimeria tenella

1994 ◽  
Vol 298 (2) ◽  
pp. 289-294 ◽  
Author(s):  
G Maga ◽  
S Spadari ◽  
G E Wright ◽  
F Focher
Keyword(s):  
Protozoan Parasite ◽  
Enzymic Activity ◽  
Eimeria Tenella ◽  
Adenosine Kinase ◽  
Guanine Nucleotides ◽  
Partial Purification ◽  
Phosphoribosyl Pyrophosphate ◽  
Phosphate Donor ◽  
Purine Salvage Pathways

From oocysts of the protozoan parasite Eimeria tenella, responsible for avian coccidiosis, we have partially purified and characterized a novel enzymic activity which specifically phosphorylates guanosine to GMP. The enzyme is able to use several phosphate donors, in the order: acetyl phosphate (Ac-P) > ATP > UTP > CTP > phosphoribosyl pyrophosphate (PRPP) > dUTP > or = dATP. The low specificity of this enzyme for the phosphate donor suggested that it be named guanosine phosphotransferase (GPTase). This enzyme is biochemically distinct from the previously described adenosine kinase (AK) and hypoxanthine/xanthine/guanine phosphoribosyltransferase (HXGPRTase), and may enable the parasite to synthesize guanine nucleotides under conditions of imbalance between adenine and guanine nucleotides. Because of its possible role in the purine salvage pathways, we have studied the effect of several guanine and guanosine analogues, recently synthesized in our laboratory, on the activity of GPTase in vitro. GPTase is specifically inhibited in the micromolar range by several substituted N2-phenylguanine bases. These results indicate that, as previously found for AK and HXGPRTase, GPTase could be a potential target for antiparasitic chemotherapy.

Toxoplasma gondii adenosine kinase: expression, purification, characterization, crystallization and preliminary crystallographic analysis

2000 ◽  
Vol 56 (1) ◽  
pp. 76-78 ◽  
Author(s):  
Rosario Recacha ◽  
Alexander Talalaev ◽  
Lawrence J. DeLucas ◽  
Debasish Chattopadhyay
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Protein ◽  
Protozoan Parasite ◽  
Adenosine Monophosphate ◽  
Crystallographic Analysis ◽  
Adenosine Kinase ◽  
Monoclinic Space Group ◽  
Cell Parameters ◽  
Ph Range

The obligate intracellular protozoan parasite Toxoplasma gondii depends on the purine-salvage pathway for its purine supply. Unlike its mammalian hosts, T. gondii salvages purine precursors predominantly via adenosine kinase, the enzyme that phosphorylates adenosine to adenosine monophosphate (AMP). The cDNA encoding T. gondii adenosine kinase was subcloned and expressed in Escherichia coli. The recombinant protein was active in an in vitro enzyme assay over a broad pH range. It required a divalent cation for activity. The enzyme was inactivated by the addition of 1 µM mercuric chloride. The inactivation could be reversed by a reducing agent. The active recombinant protein was crystallized using sodium sulfate as precipitant at pH 8.0. The crystals diffract to 1.8 Å and belong to the monoclinic space group P21, with unit-cell parameters a = 47.5, b = 68.9, c = 57.0 Å, β = 100.3°. The calculated Vm based on one molecule per asymmetric unit is 2.38 Å3 Da−1.

scholarly journals Cloning of the Schizosaccharomyces pombe gene encoding diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) asymmetrical hydrolase: sequence similarity with the histidine triad (HIT) protein family

1995 ◽  
Vol 312 (3) ◽  
pp. 925-932 ◽  
Author(s):  
Y Huang ◽  
P N Garrison ◽  
L D Barnes
Keyword(s):  
Amino Acids ◽  
Schizosaccharomyces Pombe ◽  
Sequence Similarity ◽  
Enzymic Activity ◽  
Protein Family ◽  
Partial Purification ◽  
Binding Motif ◽  
Local Similarity ◽  
Multicopy Plasmid

Diadenosine 5′,5‴-P1,P4-tetraphosphate (Ap4A) asymmetric hydrolase (EC 3.6.1.17) is a specific catabolic enzyme of Ap4A found in Schizosaccharomyces pombe. We have previously described the partial purification of Ap4A hydrolase from S. pombe [Robinson, de la Peña and Barnes (1993) Biochim. Biophys. Acta 1161, 139-148]. We determined the sequence of the N-terminal 20 amino acids of Ap4A hydrolase and designed two degenerate PCR primers based on the sequence. The 60 bp DNA fragment obtained by PCR, which is specific to Ap4A hydrolase, was used to isolate the Ap4A hydrolase gene, aph1, from S. pombe by screening a genomic DNA library in a multicopy plasmid. Ap4A hydrolase activity from the crude supernatant of a positive S. pombe transformant was about 25-fold higher than the control. There was no detectable stimulation of enzymic activity by phosphate. The aph1 gene from S. pombe contains three introns. The intron boundaries were confirmed by sequencing the cDNA of the aph1 gene from a S. pombe cDNA library. The deduced open reading frame of the aph1 gene codes for 182 amino acids. Two regions of significant local similarity were identified between the Ap4A hydrolase and the histidine triad (HIT) protein family [Séraphin (1992) DNA Sequence 3, 177-179]. HIT proteins are present in prokaryotes, yeast, plants and mammals. Their functions are unknown, except that the bovine protein inhibits protein kinase C in vitro. All four histidine residues which are conserved among the HIT proteins, including the HxHxH putative Zn(2+)-binding motif, are conserved in the Ap4A hydrolase. In addition, there are two regions of similarity between the Ap4A phosphorylases I and II from Saccharomyces cerevisiae and Ap4A hydrolase from S. pombe. These regions overlap with the HIT protein similarity regions. The aph1 gene from S. pombe is the first asymmetrical Ap4A hydrolase gene to be cloned and sequenced.

scholarly journals Partial purification and characterization of deoxyguanosine kinase from pig skin

1979 ◽  
Vol 183 (3) ◽  
pp. 547-553 ◽  
Author(s):  
F J Green ◽  
R A Lewis
Keyword(s):  
Catalytic Activity ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Nucleoside Triphosphate ◽  
Bivalent Cation ◽  
Sephadex Column ◽  
Pig Skin ◽  
Deoxyguanosine Kinase ◽  
Phosphate Donor ◽  
Ph Range

Deoxyguanosine kinase, which catalyses the phosphorylation of deoxyguanosine to form deoxyguanosine 5′-monophosphate, was purified 1024-fold from extracts to newborn-pig skin. This activity requires the presence of a bivalent cation and a nucleoside triphosphate, which functions as a phosphate donor, ATP being twice as effective as CTP or GTP and 4 times as effective as UTP. The enzyme appears to have a molecular weight of 58500 as determined by Sephadex-column chromatography. Optimal enzymic activity was observed at pH 8.0; however, the enzyme remained active over a broad pH range of 5.5-9.0. Several deoxyribonucleoside and ribonucleoside monophosphates and triphosphates were tested as effectors of catalytic activity. Effective inhibitors were dGMP [Ki(app.) = 7.6 × 10(-5) M] and dGTP [Ki(app.) = 2.1 × 10(-5) M]. Both of these inhibitors acted in a competitive manner. A Km(app.) of 3.2 × 10(-7) M was measured for deoxyguanosine and a Km(app.) of 3.3 mM was determined for MgATP. Of the four major deoxynucleosides tested, this catalytic activity appears to phosphorylate only deoxyguanosine; thus the enzyme is a specific deoxyguanosine kinase.

Binding of Tissue Plasminogen Activator to Vascular Grafts

1989 ◽  
Vol 61 (01) ◽  
pp. 131-136 ◽  
Author(s):  
Richard A Harvey ◽  
Hugh C Kim ◽  
Jonathan Pincus ◽  
Stanley Z Trooskin ◽  
Josiah N Wilcox ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Fibrinolytic Activity ◽  
Polymer Surface ◽  
Enzymic Activity ◽  
Vascular Prostheses ◽  
Chemically Modified ◽  
Insoluble Surfactant ◽  
Tissue Plasminogen

SummaryTissue plasminogen activator labeled with radioactive iodine (125I-tPA) was immobilized on vascular prostheses chemically modified with a thin coating of water-insoluble surfactant, tridodecylmethylammonium chloride (TDM AC). Surfactant- treated Dacron, polytetrafluoroethylene (PTFE), silastic, polyethylene and polyurethane bound appreciable amounts of 125I- tPA (5-30 μg 125I-tPA/cm2). Upon exposure to human plasma, the amount of 125I-tPA bound to the surface shows an initial drop during the first hour of incubation, followed by a slower, roughly exponential release with a t½ of appoximately 75 hours. Prostheses containing bound tPA show fibrinolytic activity as measured both by lysis of clots formed in vitro, and by hydrolysis of a synthetic polypeptide substrate. Prior to incubation in plasma, tPA bound to a polymer surface has an enzymic activity similar, if not identical to that of the native enzyme in buffered solution. However, exposure to plasma causes a decrease in the fibrinolytic activity of both bound tPA and enzyme released from the surface of the polymer. These data demonstrate that surfactant-treated prostheses can bind tPA, and that these chemically modified devices can act as a slow-release drug delivery system with the potential for reducing prosthesis-induced thromboembolism.

scholarly journals Dual RNA-Seq transcriptome analysis of chicken macrophage-like cells (HD11) infected in vitro with Eimeria tenella

Parasitology ◽  
2021 ◽  
Vol 148 (6) ◽  
pp. 712-725
Author(s):  
Arnar K. S. Sandholt ◽  
Feifei Xu ◽  
Robert Söderlund ◽  
Anna Lundén ◽  
Karin Troell ◽  
...  
Keyword(s):  
Transcriptome Analysis ◽  
Eimeria Tenella ◽  
Rna Seq ◽  
Chicken Macrophage

Abstract

scholarly journals Antitrypanosomal activity of purine nucleosides can be enhanced by their conversion to O-acetylated derivatives.

1996 ◽  
Vol 40 (11) ◽  
pp. 2567-2572 ◽  
Author(s):  
J R Sufrin ◽  
D Rattendi ◽  
A J Spiess ◽  
S Lane ◽  
C J Marasco ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Parent Compound ◽  
Nucleoside Analogs ◽  
Significant Activity ◽  
Structural Class ◽  
Antitrypanosomal Activity ◽  
Salvage Pathways ◽  
Purine Salvage Pathways

Fifteen purine nucleosides and their O-acetylated ester derivatives were examined for in vitro antitrypanosomal activity against the LAB 110 EATRO isolate of Trypanosoma brucei brucei and two clinical isolates of Trypanosoma brucei rhodesiense. Initial comparisons of activity were made for the LAB 110 EATRO isolate. Three nucleoside analogs exhibited no significant activity (50% inhibitory concentrations [IC50s] of > 100 microM), whether they were O acetylated or unacetylated; three nucleosides showed almost equal activity (IC50s of < 5 microM) for the parent compound and the O-acetylated derivative; nine nucleosides showed significantly improved activity (> or = 3-fold) upon O acetylation; of these nine analogs, six displayed activity at least 10-fold greater than that of their parent nucleosides. The most significant results were those for four apparently inactive compounds which, upon O acetylation, displayed IC50s of < or = 25 microM. When the series of compounds was tested against T. brucei rhodesiense isolates (KETRI 243 and KETRI 269), their antitrypanosomal effects were comparable to those observed for the EATRO 110 strain. Thus, our studies of purine nucleosides have determined that O acetylation consistently improved their in vitro antitrypanosomal activity. This observed phenomenon was independent of their cellular enzyme targets (i.e., S-adenosylmethionine, polyamine, or purine salvage pathways). On the basis of our results, the routine preparation of O-acetylated purine nucleosides for in vitro screening of antitrypanosomal activity is recommended, since O acetylation transformed several inactive nucleosides into compounds with significant activity, presumably by improving uptake characteristics. O-acetylated purine nucleosides may offer in vivo therapeutic advantages compared with their parent nucleosides, and this possibility should be considered in future evaluations of this structural class of trypanocides.

scholarly journals Localization of ferrochelatase in Plasmodium falciparum

2004 ◽  
Vol 384 (2) ◽  
pp. 429-436 ◽  
Author(s):  
Sundaramurthy VARADHARAJAN ◽  
B. K. Chandrashekar SAGAR ◽  
Pundi N. RANGARAJAN ◽  
Govindarajan PADMANABAN
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Enzymic Activity ◽  
Malarial Parasite ◽  
Parasite Genome ◽  
Marker Proteins ◽  
Parasite Plasmodium ◽  
Theoretical Predictions ◽  
Parasite Plasmodium Falciparum

Our previous studies have demonstrated de novo haem biosynthesis in the malarial parasite (Plasmodium falciparum and P. berghei). It has also been shown that the first enzyme of the pathway is the parasite genome-coded ALA (δ-aminolaevulinate) synthase localized in the parasite mitochondrion, whereas the second enzyme, ALAD (ALA dehydratase), is accounted for by two species: one species imported from the host red blood cell into the parasite cytosol and another parasite genome-coded species in the apicoplast. In the present study, specific antibodies have been raised to PfFC (parasite genome-coded ferrochelatase), the terminal enzyme of the haem-biosynthetic pathway, using recombinant truncated protein. With the use of these antibodies as well as those against the hFC (host red cell ferrochelatase) and other marker proteins, immunofluorescence studies were performed. The results reveal that P. falciparum in culture manifests a broad distribution of hFC and a localized distribution of PfFC in the parasite. However, PfFC is not localized to the parasite mitochondrion. Immunoelectron-microscopy studies reveal that PfFC is indeed localized to the apicoplast, whereas hFC is distributed in the parasite cytoplasm. These results on the localization of PfFC are unexpected and are at variance with theoretical predictions based on leader sequence analysis. Biochemical studies using the parasite cytosolic and organellar fractions reveal that the cytosol containing hFC accounts for 80% of FC enzymic activity, whereas the organellar fraction containing PfFC accounts for the remaining 20%. Interestingly, both the isolated cytosolic and organellar fractions are capable of independent haem synthesis in vitro from [4-14C]ALA, with the cytosol being three times more efficient compared with the organellar fraction. With [2-14C]glycine, most of the haem is synthesized in the organellar fraction. Thus haem is synthesized in two independent compartments: in the cytosol, using the imported host enzymes, and in the organellar fractions, using the parasite genome-coded enzymes.

Effect of the Probiotic Lactobacillus Plantalum CMU-FP002 on Oocyst Shedding by Broilers Inoculated with Eimeria Tenella

2009 ◽  
Vol 2 (3) ◽  
pp. 157-159 ◽  
Author(s):  
Charunee Kasornpikul ◽  
Chaiyavat Chaiyasut ◽  
Bussabun Sirithanyalug ◽  
Worapol Aeagwanich ◽  
Thanit Pewnim
Keyword(s):  
Infectious Disease ◽  
Health Problem ◽  
Protozoan Parasite ◽  
Eimeria Tenella ◽  
Control Group ◽  
Post Inoculation ◽  
Inoculum Dose ◽  
Probiotic Lactobacillus ◽  
Reported Health ◽  
Genus Eimeria

Coccidiosis is an infectious disease that causes the most widespread health problems in the broiler industry. This study indicated that chickens fed with the probiotic Lactobacillus plantalum CMU- FP002 exhibited an 85.63% reduction in the number of oocysts of Eimeria tenella shed in the faeces compared with the control group. The average number of oocysts shed by the group fed the probiotic and a group fed an antibiotic were both significantly (P<0.05) less than the average number of oocysts shed by the control group. Coccidiosis is an infectious disease caused by a protozoan parasite of the genus Eimeria and is the most consistently reported health problem in poultry (Biggs, 19–82; Rose et al, 1987; Williams, 1999). Generally, the number of oocysts shed in the faeces is dependent on the number of sporozoites and merozoites that penetrate the enterocytes for a given inoculum dose, and so represents the infection-resistant ability of broilers. Susceptibility to Eimeria can also be assessed on the basis of the number of oocysts obtained from droppings collected for four days starting on day 6 post-inoculation (Dalloul et al., 2005).

scholarly journals All1371 is a polyphosphate-dependent glucokinase in Anabaena sp. PCC 7120

Microbiology ◽  
2014 ◽  
Vol 160 (12) ◽  
pp. 2807-2819 ◽  
Author(s):  
Friederike Klemke ◽  
Gabriele Beyer ◽  
Linda Sawade ◽  
Ali Saitov ◽  
Thomas Korte ◽  
...  
Keyword(s):  
Divalent Cations ◽  
Enzymic Activity ◽  
Phosphoryl Group ◽  
Nitrogen Deprivation ◽  
Ping Pong ◽  
Nucleoside Triphosphates ◽  
Anabaena Sp ◽  
Pcc 7120 ◽  
Promoter Studies

The polyphosphate glucokinases can phosphorylate glucose to glucose 6-phosphate using polyphosphate as the substrate. ORF all1371 encodes a putative polyphosphate glucokinase in the filamentous heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. Here, ORF all1371 was heterologously expressed in Escherichia coli, and its purified product was characterized. Enzyme activity assays revealed that All1371 is an active polyphosphate glucokinase that can phosphorylate both glucose and mannose in the presence of divalent cations in vitro. Unlike many other polyphosphate glucokinases, for which nucleoside triphosphates (e.g. ATP or GTP) act as phosphoryl group donors, All1371 required polyphosphate to confer its enzymic activity. The enzymic reaction catalysed by All1371 followed classical Michaelis–Menten kinetics, with k cat = 48.2 s−1 at pH 7.5 and 28 °C and K M = 1.76 µM and 0.118 mM for polyphosphate and glucose, respectively. Its reaction mechanism was identified as a particular multi-substrate mechanism called the ‘bi-bi ping-pong mechanism’. Bioinformatic analyses revealed numerous polyphosphate-dependent glucokinases in heterocyst-forming cyanobacteria. Viability of an Anabaena sp. PCC 7120 mutant strain lacking all1371 was impaired under nitrogen-fixing conditions. GFP promoter studies indicate expression of all1371 under combined nitrogen deprivation. All1371 might play a substantial role in Anabaena sp. PCC 7120 under these conditions.

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