Thymidylate synthetase during synchronous nuclear division cycle and differentiation of Physarum polycephalum

Thymidylate synthetase and thymidine kinase activities in wild type strain M₃b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M₃b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism.     

Study of Analogues of Thymidine-5′-Monophosphate and Thymidine as Substrates or Inhibitors of Chick Embryo Liver Thymidylate Kinase

Abstract

The phosphorylation of thymidine-5′-monophosphate (dTMP) by chick embryo liver thymidylate kinase (Km (dTMP) =1.2 μM) was inhibited by the 5′-monophosphate derivatives of 5-bromo-2′-deoxyuridine (5-Br-dUMP), 5-iodo-2′-deoxyuridine (5-I-dUMP), 2′,3′-dideoxythymidine (ddTMP), 3′-azido-3′-deoxythymidine (AZT-MP) and the methylene phosphonate analogue of AZT-MP with IC50 values of 8, 24, 14, 5 and 6 μM respectively. 5-Fluoro-2′-deoxyuridine (5-F-dUMP) and dUMP were poor inhibitors (IC50 values > 300 μM). 5-Br-dUMP and 5-I-dUMP were found to be significant substrates of thymidylate kinase with phosphorylation efficiencies (Vmax/Km) of 26 and 6% of that of dTMP, respectively. In contrast, AZT-MP and ddTMP were poor substrates, being phosphorylated 800-fold less efficiently than dTMP. Thymidylate kinase was also significantly inhibited by thymidine and AZT. Our data give a better insight into the topology of the dTMP binding site of this enzyme and show that the 3′-hydroxyl group of dTMP plays a critical role in catalysis.     

Identification of a developmentally regulated sialidase inEimeria tenella that is immunologically related to theTrypanosoma cruzi enzyme

Sporozoites and merozoites of three species ofEimeria, E. tenella, E. maxima, andE. necatrix, that cause diarrhea in chickens worldwide, were examined for their expression of sialidase (SA) activity. The enzyme was found in three species, and the activity of merozoites was 10–20 times higher than that of sporozoites. The enzyme was resistant to degradation by proteases that are normally present in the intestine, a site inhabited by theEimeria parasites, and it was relatively resistant to heat, with optimum activity being at 40°C, which is within the range of temperature in the chicken intestine (40–43°C).E. tenella SA was immuniprecipitated by monoclonal and polyclonal antibodies raised against theTrypanosoma cruzi SA (TCSA), and enzyme activity was neutralized by these antibodies.E. tenella SA was identified by immunoblots as a doublet of molecular weight 190 000 and 180 000 using, as a probe, anti-TCSA antibodies and antibodies against a synthetic peptide (TR) derived from the long tandem repeat domain of TCSA. Binding of the monoclonal and polyclonal antibodies toE. tenella was completely blocked by TR, but not by an irrelevant peptide (BR). Therefore,E. tenella expresses a developmentally regulated SA that is structurally related to theT. cruzi counterpart. Because of the high SA activity in merozoites, and by analogy with other SA-producing microbes that inhabit mucin-rich epithelia, we suggest that theEimeria SA plays a role in desialylating intestinal mucins to reduce viscosity of the local environment and thereby facilitate parasite migration. The enzyme could also play a role in host cell-parasite interaction.Abbreviations SA sialidase (neuraminidase) - Neu5Ac N-acetylneuraminic acid - 4-MU-Neu5Ac 2-(4-methylumbelliferyl)--N-acetyl-d-neuraminic acid - BSA bovine serum albumin - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PNA peanut agglutinin - Ab antibody - TCN-2 monoclonal antibody toT. cruzi sialidase, anti-Ars, monoclonal antibody top-azophenylarsonate - TCSA Trypanosoma cruzi sialidase     

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene.     

Localization of Some Enzymes of β-oxidation of Fatty Acids in the Peroxisomes of Tetrahymena*

Mitochondria and peroxisomes were prepared from homogenates of Tetrahymena pyriformis by sedimentation through sucrose gradients. Catalase and isocitrate lyase served as peroxisomal markers; lactic dehydrogenase and glutamic dehydrogenase as mitochondrial markers. Acetyl-CoA synthetase, octanoyl-CoA synthetase, palmitoyl-CoA synthetase, 3-β hydroxyacyl CoA dehydrogenase, and thiolase activities were found in both the peroxisomes and the mitochondria. It is suggested that β-oxidation of fatty acids accurs in both organelles in Tetrahymena.     

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant Plasmodium chabaudi

Alteration in properties of thymidylate synthetase from pyrimethamine-resistant smodium chabaudi. International Journal for Parasitology16: 483–490. Thymidylate synthetase from cloned strains of pyrimethamine-sensitive and resistant P. chubaudi were partially purified and characterized. The enzyme from both strains have equal mol. wt of 120,000 as estimated by Sephadex G-200 column chromatography. The enzyme from drug-sensitive parasites has an optimum pH of 6.5–7.5 and is stable at pH 4–11 while that from drug-resistant strain has an pH optimum of 7.0–8.0 and is stable at pH 5–10. The K_m for methylenetetrahydrofolate are 206 ± 6 and 495 ± 5 μm for the enzyme from drug-resistant and sensitive parasites, respectively. The K_m for dUMP of the enzyme from drug-resistant and sensitive parasites are 42 ± 1 and 49 ± 6 μm, respectively. Inhibition of the enzyme from both strains by FdUMP are competitive with dUMP; however,the K_is for the enzyme from drug-resistant strain (0.043 ± 0.005 μm) is less than that from drug-sensitive strain (0.11 ± 0.007 μm) by a factor of 2.5. The K_ii for methotrexate with respect to methylenetetrahydrofolate of the enzyme from drug-resistant parasites (58 ± 3 μm) is 3 times larger than that from drug-sensitive parasites (17 ± 1 μm).     

Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei

Pattanakitsakul S. and Ruenwongsa P. 1984. Characterization of thymidylate synthetase and dihydrofolate reductase from Plasmodium berghei. International Journal for Parasitology14: 513–520. Thymidylate synthetase (TS) and dihydrofolate reductase (DHFR) from Plasmodium berghei were copurified by Sephacryl S-300 and Sephadex G-200 column chromatography and found to have an apparent mol. wt of 132,000. Electrophoresis of the partially purified enzyme under non-denaturing conditions showed the comigration of TS and DHFR. The mol. wt of TS was estimated to be 65,000 on SDS-gel electrophoresis. Both enzymes exhibit a broad pH optimum in the range of 6.5–8.0. Urea, NaCl and KC1 inhibit TS but activate DHFR. For TS, the apparent K_m for dUMP and methylene-tetrahydrofolate have been found to be 71.4 and 312.5 μM, respectively. For DHFR, the apparent K_m for dihydrofolate and NADPH have been found to be 4.4 and 12.5 μM, respectively. Inhibition of DHFR by pyrimethamine, methotrexate and trimethoprim are competitive with dihydrofolate with K_is of 0.63, 0.5 and 1.88 nM, respectively. FdUMP inhibition of TS is competitive with dUMP with K_is of 0.05 μM, but inhibition by methotrexate is uncompetitive with dUMP and MTHF with K_ii of 103 and 23 μM, respectively.     

Isolation and lipid composition of nuclear membranes from macronuclei of Tetrahymena pyriformis

A procedure was developed for isolation of macronuclei and nuclear membranes from the ciliated protozoan Tetrahymena pyriformis E, and the lipid composition of the isolated nuclear membranes was determined.This method involves cell lysis with octanol, separation of the nuclear membrane with 0.2 M phosphate–1M NaCl and purification on a discontinuous sucrose gradient. By phase-contrast and electron microscopic examinaton, our preparations were pure and preserved the typical nuclear membrane morphology: inner and outer nuclear membranes, and nuclear pore complexes. As for lipid distribution, the three major phospholipids in the membranes were phosphatidylcholine (31.0%), phosphatidylethanolamine (26.1%) and 2-aminoethylphosphonolipids (23.3%) and the molar ratio of a sterol-like lipid, tetrahymanol to phospholipid phosphorus was 0.036. These results were compared to other membrane fractions of Tetrahymena.     

Purification and Characterization of Phospholipase C Preferentially Hydrolysing Phosphatidylcholine in Tetrahymena Membranes

A phospholipase C (PLC) activity that preferentially hydrolyses phosphatidylcholine to diacylglycerol and phosphorylcholine was found to be present in Tetrahymena pyriformis, strain W and most of its activity was recovered in the membrane fraction. This enzyme was extracted with 1% Triton X-100 from the membrane fraction and purified to apparent homogeneity by sequential chromatographies on Fast Q-Sepharose, hydroxyapatite HCA-100S, Mono Q and Superose 12 gel filtration columns. The purified enzyme had specific activity of 2083 nmol of diacylglycerol released/mg of protein/min for dipalmitoylphosphatidylcholine hydrolysis. Its apparent molecular mass was 128 kDa as determined by SDS-polyacrylamide gel electrophoresis and was 127 kDa by gel filtration chromatography, indicating that the enzyme is present in a monomeric form. The enzyme exhibited an optimum pH 7.0 and the apparent K_m value was determined to be 166 μM for dipalmitoylphosphatidylcholine. A marked increase was observed in phosphatidylcholine hydrolytic activity in the presence of 0.05% (1.2 mM) deoxycholate. Ca²⁺ but not Mg²⁺ enhanced the activity at a concentration of 2 mM. This purified phospholipase C exhibited a preferential hydrolytic activity for phosphatidylcholine but much less activity was observed for phosphatidylinositol (～ 9%) and phosphatidylethanolamine (～ 2%).     

Properties and inhibition of thymidylate synthetase in Drosophila melanogaster

A quantitative comparison of the incorporation of methyl-³H-thymidine and 6-³H-deoxyuridine into the DNA of Drosophila melanogaster in the presence and in the absence of 5-fluorouracil indicated that 5-fluorouracil inhibits the reaction converting dUMP to dTMP catalysed by thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, E.C. 2.1.1.b). The enzyme exhibits maximal activity at pH 7·5 to 8·0 and is protected from heat inactivation by deoxyuridine monophosphate. The addition of thiol compounds to the homogenization buffer results in the enhancement of synthetase activity. The K_m values for deoxyuridine monophosphate and 5,10-methylenetetrahydrofolate are 6·8 × 10^?6 M and 8·3 × 10^?5 M, respectively. Fluorodeoxyuridine monophosphate, trifluoromethyldeoxyuridine monophosphate, and methotrexate are inhibitors of the enzyme. 5-Bromodeoxyuridine and 5-iododeoxyuridine have no inhibitory effect. The results support the contention that, under conditions which induce morphological lesions in Drosophila, fluorinated pyrimidines and methotrexate inhibit the de novo synthesis of thymidylate whereas thymidine analogues function in some other manner.     

1-O-Alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) is a minor lipid component in Tetrahymena pyriformis cells

The protozoan Tetrahymena pyriformis contains 4.2 ± 2.2 ng PAF/10⁷ cells. Only 1–3% of this lipid is released in the cell free medium. PAF production is not influenced by different extracellular Ca²⁺ concentrations. Cell stimulation with calcium ionophore A23187 or zymosan particles does not affect the amount of PAF either. This is the first report of a natural occurrence of PAF in a protozoan.     

Properties of Purified L-Ornithine Decarboxylase (EC 4.1.1.17) from Tetrahymena thermophila

Ornithine decarboxylase (ornithine carboxy lyase; EC 4.1.1.17) (ODC) from Tetrahymena thermophila was purified 6,300 fold employing fractionated ammonium sulfate precipitation, gel permeation chromatography on Sephadex G-150, ion exchange chromatography on DEAE-Sepharose CL-6B, and preparative isoelectric focussing. The product obtained in 24% yield was a preparation of the specific activity of 10,200 nmol CO₂mdh^-1mdmg^-1. The purified enzyme was rather stable at 37°C (14% loss of activity within 1 h). The molecular and catalytic properties of this enzyme were investigated. The isoelectric point was 5.7 and the molecular weight (MW) was estimated to be 68,000 under nondenaturing conditions. The pH optimum was between 6.0 and 7.0, the K_m for the substrate L-ornithine was 0.11 mM, and the K_m for the cofactor pyridoxal 5-phosphate was 0.12 μM; the product of ODC catalysis, putrescine, was a poor inhibitor with an estimated K_i of about 10 mM. The enzyme was inhibited competitively by D-ornithine with a K_i of 1.6 mM and by α-difluoromethylornithine with a K_i of 0.15 mM. The latter one, an enzyme activated irreversible inhibitor of mammalian ODC, inactivated the enzyme from T. thermophila at high concentrations with a half life time of 14 min. Other basic amino acids, e.g. L-lysine, L-arginine, and L-histidine, were neither substrates nor inhibitors of the enzyme, as were the diamines 1,3-diaminopropanol and cadaverine, the polyamines spermidine and spermine and the cosubstrate analogues pyridoxal and pyridoxamine-5-phosphate. Polyanions were activators of the enzyme: The half maximal ODC stimulating concentrations were 2.2 μgmdml^-1 for RNA, 6.1 μgmdml^-1 for DNA, and 0.25 μgmdml^-1 for heparin. These results indicate that ODC from T. thermophila shares several properties with ODC preparations from other organisms but in some respects, especially in activator and inhibitor specificity, there are some special qualities unique to this particular protozoan enzyme.     

Virus-like particles in Eimeria tenella are associated with multiple RNA segments

Total nucleic acids from sporulated oocysts of Eimeria tenella isolated from Changchun in China were found to contain three extrachromosomal double-stranded RNA segments (dsRNAs) of 1.4, 2.4 and 3.6 kb in sizes. These RNAs were resistant to RNase A digestion under high salt concentration (0.3 M NaCl). RNA-dependent RNA polymerase (RDRP) activity was detected in crude extracts of E. tenella sporulated oocysts containing these nucleic acid species. Virus-like particles (VLPs) were shown to have a diameter of approximately 38 nm under Electron Microscopy (EM) after purification by sucrose density gradient centrifugation. In keeping with the nomenclature generally adopted for protozoan viruses, we have named this isolate as E. tenella virus (ETV) which is the first virus isolated from E. tenella.     

An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.     

Inhibition of conjugation and cell division in Tetrahymena pyriformis by tunicamycin: A possible requirement of glycoprotein synthesis for induction of conjugation

Tunicamycin, a glucosamine-containing antibiotic inhibited the conjugation process of Tetrahymena pyriformis. Sexual pairing was prevented completely when 1.5 μg/ml of tunicamycin was added to a mixture of the two mating types. Tunicamycin caused preferential inhibition of glycoprotein synthesis in Tetrahymena pyriformis. At 1.5 μg/ml and 6 μg/ml tunicamycin inhibited by 40% and 60% respectively [³H]-glucosamine incorporation into material precipitated by ethanol, while it did not affect [¹⁴C]-leucine incorporation. Cell division was also inhibited when the drug was added either to the regular growth medium or to the starvation medium.     

L-asparaginase of Tetrahymena pyriformis is associated with a kinase activity

Most of L-asparaginase activity of Tetrahymena pyriformis was found to be present in microsomal membranes from which it has been purified to homogeneity (Tsirka, S.A.E. and Kyriakidis, D.A. Mol. Cell. Biochem. 83: 147–155, 1988). The native enzyme has a relative molecular weight of approximately 200 kDa, while under denaturing conditions the enzyme exhibits. a subunit size of 39 kDa. Aminoacid analysis and an oligopeptide from N-terminal sequence have been determined. Dephosphorylation of L-asparaginase by alkaline phosphatase results in an activation of its catalytic activity. This enzyme also exhibits intrinsic phosphorylation activity with a Km value for ATP of 0.5 mM. Autophosphorylation with -^32P ATP of purified L-asparaginase results in the phosphorylation of tyrosine residues as well as in loss of its activity. Mg²⁺ and Ca²⁺ added together act synergistically to stimulate the kinase activity by more than 160%. The polyamines putrescine, spermidine and spermine activate the kinase approximately 100%, while neither cAMP or cGMP have any effect. These results indicate that this membrane protein with dual L-asparaginase/kinase activity must play an important role in regulating the intracellular levels of L-asparagine in Tetrahymena pyriformis.     

5′-Haloacetamido-5′-deoxythymidines: novel inhibitors of thymidylate synthase

5′-Bromoacetamido-5′-deoxythymidine (BAT), 5′-iodoacetamido-5′-deoxythymidine (IAT), 5′-chloroacetamido-5′-deoxythymidine (CAT) and [¹⁴C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were invesatigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT > IAT > CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4–8 fold higher than it was in the absence of dUMP. The I₅₀ values for BAT were 1·10⁻⁵ M and 1.2·10⁻⁶ M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thynmidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The K_m for the enzyme was 9.2 μM, and the K_i determined for competitive inhibition by BAT was 5.4 μM. Formation of a tight, irreversible compledx is referred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min⁻¹, and the steady-state constant of inactivation, K_i, was estimated to be 6.6 μM. The 5′-haloacetamido-5′-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.     

Itaconic acid: An inhibitor of isocitrate lyase in Tetrahymena pyriformis in vitro and in vivo

The succinate analog itaconic acid was observed to be a competitive inhibitor of the glyoxylate cycle specific enzyme isocitrate lyase (EC 4.1.3.1) in cell-free extracts of Tetrahymena pyriformis. Itaconic acid also inhibited net in vivo glycogen synthesis from glyoxylate cycle-dependent precursors such as acetate but not from glyoxylate cycle-independent precursors such as fructose. The effect of itaconic acid on the incorporation of ¹⁴C into glycogen from various ¹⁴C-labeled precursors was also consistent with inhibition of isocitrate lyase by this compound. Another analog of succinate which shares a common metabolic fate with itaconic acid, mesaconic acid, had no effect on isocitrate lyase activity in vitro or on ¹⁴C-labeled precursor incorporation into glycogen in vivo. In addition, itaconic acid did not affect gluconeogenesis from lactate in isolated perfused rat livers, a system lacking the enzyme isocitrate lyase. These results are taken as evidence that itaconic acid is an inhibitor of glyoxylate cycle-dependent glyconeogenesis Tetrahymena pyriformis via specific competitive inhibition of isocitrate lyase activity.     

Partial purification and characterization of thymidylate kinase from embryonic chick liver.

Thymidylate kinase from the livers of 18-day-old chick embryos was concentrated 423-fold. The purification procedure included acid precipitation, ammonium sulfate fractionation, gel filtration on Sephadex G-100 and G-75 Super Fine, and ion-exchange chromatography on Diethylaminoethyl Sephadex A-50. This enzyme was found to be very labile but could be stabilized for long periods of time by its substrate (thymidine 5′-monophosphate) in the presence of 2-mercaptoethanol. Enzymes responsible for the formation of thymidine 5′-diphosphate and thymidine 5′-triphosphate, respectively, were separated during fractionation procedures. Thymidylate kinase from chick embryo liver was found to be a single protein having a molecular weight of approximately 46,000, Michaelis constant approximately 8 × 10^?5m, and a broad pH optimum between 6.6 and 8.6. A 2–3 mm requirement of Mg²⁺ above the adenosine 5′-triphosphate concentration was shown to be necessary for maximum enzyme activity. The enzyme appears to be competitively inhibited by thymidine, thymidine 5′-diphosphate, and thymidine 5′-triphosphate and noncompetitively inhibited by adenosine 5′-diphosphate.Thymidylate kinase enzymes isolated from two stages of developing embryonic liver and adult chick liver were shown to be identical.