Characterization of an immunodominant Onchocerca volvulus antigen with patient sera and a monoclonal antibody.

Adult Onchocerca voluvlus and infective larvae, but not microfilariae contain an immunodominant antigen (33,000 and 21,000 Mr in females, 39,000, 33,000, and 21,000 Mr in males, 133,000 Mr in infective larvae) which is recognized by an Onchocerca-specific mAb. The component is part of the reproductive organs and muscles. 96.2% of onchocerciasis sera contained antibodies detectable by immunoblotting against it. Antigen purified by immunoaffinity chromatography was specifically recognized in immunoblots by onchocerciasis sera, but not by sera from other filarial infections. The high immunogenicity, the specificity, and the occurrence in infective larvae of this antigen indicate an immunodiagnostic potential and a possible role in the immunobiology of the parasite.

Nucleoside-dependent protein kinase from Trypanosoma gambiense.

A nucleoside-dependent protein kinase (EC 2.7.1.37) was partially purified from Trypanosoma gambiense, the pathogenic agent of sleeping sickness. This enzyme catalyzes the phosphorylation of histone and protamine. Various nucleosides at the concentration of 10(-4) M stimulated the histone kinase activity about two-fold, whereas cyclic AMP and cyclic GMP were without effect. The pH-optimum for histone phosphorylation was at about pH 7.0. The enzyme activity absolutely depends on Mg2+, Mn2+ or Co2+. The apparent Km-value for histone was 0.3 mg/ml and those for ATP were 2 - 10(-4) M and 6 - 10(-5) M in the absence or presence of 10(-4) M adenosine respectively. IDP and ADP complete with ATP. The inhibition constants were calculated to be 2 - 10(-4) M and 2.5 - 10(-4) M, respectively. The molecular weight of the histone kinase was found to be 95 000 by gel filtration and 88 000 by sedimentation in a sucrose gradient.

Targeting polyamines of parasitic protozoa in chemotherapy.

All parasitic protozoa contain polyamines and in recent years they, and their associated enzymes, have attracted attention as drug targets because they might reveal novel antiparasite therapies. How justified is this approach to drug discovery? In this review, Sylke Müller, Graham Coombs and Rolf Walter summarize the current status of research into drugs that exploit polyamine metabolism of trypanosomatid and malaria parasites, and propose priorities for research into such drugs. This review was inspired by an Expert Meeting entitled 'Polyamine Metabolism of Parasitic Protozoa as a Drug Target'.

Call to consolidate achievements for onchocerciasis and lymphatic filariasis control.

The Bernhard Nocht Institute for Tropical Medicine and the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases held an international conference to review recent achievements in research and control of onchocerciasis and lymphatic filariasis on 19-23 September 2001 in Hamburg, Germany.

The role of the C-terminus for catalysis of the large thioredoxin reductase from Plasmodium falciparum.

The thioredoxin system is one of the major thiol reducing systems of the cell. Recent studies have revealed that Plasmodium falciparum and human thioredoxin reductase represent a novel class of enzymes, which are substantially different from the isofunctional prokaryotic Escherichia coli enzyme. We identified the cysteines Cys88 and Cys93 as the redox active disulfide and His509 as the active site base [Gilberger, T.-W., Walter, R.D. and Müller, S., J. Biol. Chem. 272 (1997) 29584-29589]. In addition to the active site thiols Cys88 and Cys93 the P. falciparum enzyme has another pair of cysteines at the C-terminus: Cys535 and Cys540. To assess the possible role of these peripheral cysteines in the catalytic process the single mutants PfTrxRC535A and PfTrxRC540A, the double mutant P/TrxRC535AC540A and the deletion mutant PfTrxRdelta9 (C-terminal deletion of the last nine amino acids) were constructed. All mutants are defective in their thioredoxin reduction activity, although they still show reactivity with 5,5'-dithiobis (2-nitrobenzoate). These data imply that the C-terminal cysteines are crucially involved in substrate coordination and/or electron transfer during reduction of the peptide substrate.

Evoked response and after-discharge thresholds to electrical stimulation in temporal lobe epileptics.

The records of electrical stimulation of 30 psychomotor epileptics were examined for threshold levels of evoked responses and after-discharge production. Comparisons were made between anatomical sites, operated and nonoperated structures, and different disease states. It was found that patients with hippocampal sclerosis (HS) had higher thresholds in structures on the operated side. The patients with lesions other than HS were found to differ from those with hippocampal sclerosis in that there were no differences found between their diseased and nondiseased structures. For both patient groups, the amygdala had a much higher threshold for after-discharge production than either the hippocampus or the hippocampal gyrus.

Altered dihydrofolate reductase in pyrimethamine-resistant Plasmodium falciparum.

Dihydrofolate reductase (EC 1.5.1.3, tetrahydrofolate dehydrogenase), the target enzyme for the chemotherapeutic attack by pyrimethamine, has been studied in drug-sensitive and resistant strains of Plasmodium falciparum. No evidence was found for overproduction of this enzyme in drug-resistant strains. Results presented here indicate that pyrimethamine resistance of P. falciparum depends on a modified dihydrofolate reductase, which shows less affinity for pyrimethamine and dihydrofolate. The inhibition constants for pyrimethamine increased from 0.19 nM for the drug-sensitive strain FCH-5 to 4.1 and 21.6 nM for the drug-resistant strains FVOR and K 1, respectively. In addition, the Km-values for dihydrofolate increased from 2.5 microM to 21 and 28 microM, respectively. The type of inhibition by pyrimethamine changed from competitive with respect to dihydrofolate in drug-sensitive strain to non-competitive in drug-resistant strains of P. falciparum.

Effect of suramin on phosphorylation-dephosphorylation reactions in Trypanosoma gambiense.

Suramin which is used as a chemotherapeutic agent against sleeping sickness was tested for its ability to inhibit phosphorylation-dephosphorylation reactions in Trypanosoma gambiense. Protein-kinase I, which is characterized by its preference for acidic acceptor proteins, was found to be strongly inhibited by suramin. The inhibition constant was calculated to be 0.8 microM. The type of inhibition was non-competitive with respect to ATP as well as to substrate protein.

Purification and characterization of gamma-glutamylcysteine synthetase from Ascaris suum.

We have purified and characterized the Ascaris suum gamma-glutamylcysteine synthetase, the rate-limiting step in the glutathione biosynthesis. The purified enzyme exhibited a specific activity of 18 U (mg protein)-1. Estimation of the molecular mass of the native enzyme by FPLC on Superdex S-200 revealed the presence of two enzyme activity peaks corresponding to molecular masses of 100 and 70 kDa. The higher-molecular-mass component could be dissociated by repeated gel filtration into the 70-kDa protein which is the enzymatically active subunit. The apparent Km values of the A. suum enzyme for L-aminobutyrate, L-cysteine and L-glutamate were 0.31, 0.41 and 0.94 mM, respectively. D,L-Buthionine-S,R-sulfoximine and cystamine showed time-dependent irreversible inhibitory effects on the A. suum enzyme activity with Ki values of 0.05 and 1.11 microM, respectively. The Ki values for the corresponding enzyme from rat kidney with D,L-buthionine-S,R-sulfoximine and cystamine were 7.19 and 22.2 microM, respectively. The time of half-inactivation of the enzyme at infinite concentration of D,L-buthionine-S,R-sulfoximine, tau 50, was determined to be 3.1 and 1.34 min, for the parasite and mammalian enzymes respectively. For cystamine, a tau 50 value of 3.32 min for the A. suum gamma-glutamylcysteine synthetase was determined, while a value of 2 min in case of rat kidney enzyme was found. The A. suum enzyme activity was competitively inhibited by glutathione with a Ki value of 0.11 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Polyamine N-acetyltransferase from Fasciola hepatica.

A cytosolic polyamine N-acetyltransferase which catalyses polyamine and diamine acetylation has been partially purified from the liver fluke Fasciola hepatica. The enzyme has an apparent Mr of 50,000 and unlike the corresponding mammalian liver counterpart is capable of putrescine acetylation. Among the substrates tested, spermidine had the highest reaction rate but putrescine had a lower Km value. The Km values for spermidine, spermine, norspermidine, putrescine, cadaverine and 1,3-diaminopropane were 20 microM, 1.30 mM, 20 microM, 7 microM, 10 microM and 50 microM, respectively. Acetylated polyamines were also substrates for the trematode acetylase, but histones were inactive. The partially purified enzyme had no deacetylase activity. The Km for acetyl-CoA was 4.4 microM. Coenzyme A was strongly inhibitory with a Ki value of 5.3 microM. Bis(benzyl)polyamine analogue MDL 27695 was a potent competitive inhibitor of the enzyme with a Ki of 22 microM. Inhibition by 1,4-dimethyl-putrescine was non-competitive and had a Ki value of 15 microM. The trematode acetylase is highly dependent on sulfhydryl groups for its activity. As had been reported in nematodes, polyamine acetylation could represent a process by which trematodes convert excess polyamines to forms suitable for transport and excretion. On the other hand, this could be the regulatory step of a functional interconversion pathway in these parasites.

Purification and characterization of gamma-glutamyl transpeptidase from Ascaris suum.

gamma-Glutamyl transpeptidase, which is of central importance in the degradation of glutathione, was purified from Ascaris suum to apparent homogeneity. The enzyme was found to be a predominantly membrane-bound protein and was solubilized by Triton X-100. The purified enzyme, which exhibits a specific activity of 1009 U (mg protein)-1, showed a molecular mass of 70 kDa and was found to be composed of two non-identical subunits of molecular mass 43 and 30 kDa. Concerning the kinetic properties of the enzyme, the data presented in this study showed that various amino acids and dipeptides with L-configuration served as acceptors for the gamma-glutamyl moieties of the enzyme reaction products and showed Km-values in the mM range. The apparent Km-value for the gamma-glutamyl donor L-glutamyl-gamma-7-amido-4-methylcoumarin of the enzyme was found to be 0.03 mM. L- and D-serine in combination with borate ions were competitive inhibitors of the enzyme activity with Ki-values of 0.30 and 0.61 mM, respectively. Acivicin was an irreversible inhibitor of the enzyme with a Ki-value of 0.42 mM and with a pseudo-first-order kinetics (kinact) of 0.18 min-1. In vitro treatment of the adult A. suum with acivicin resulted in a dose-dependent inhibition of the enzyme activity and an increase of the glutathione levels. These findings indicate the physiological role of the gamma-glutamyl transpeptidase of this parasitic nematode in the catabolism of glutathione.

Cyclic AMP-dependent and independent protein kinases in Ascaridia galli.

The occurrence of multiple protein kinases, distinguished with respect to molecular weight and preference for acceptor proteins, was demonstrated in Ascaridia galli. The molecular weights of the cyclic AMP-dependent protein kinase and of phosvitin kinase I and II - both independent of cyclic AMP-were determined to be 160000, greater than 200000 and 40000, respectively. The cyclic AMP-dependent protein kinase preferred histones and kemptide as acceptor substrates; stimulation of enzyme activity was up to 4-fold by cyclic AMP. The activities of phosvitin kinase I and II were found to be effectively inhibited by suramin. The inhibition constants were calculated to be 2 microM and 5 microM, respectively. In addition, stibophen turned out to be a potent inhibitor of phosvitin kinase I; the inhibition constant was determined to be 10 microM.

Subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in Trypanosoma brucei.

The subcellular distribution of adenylate cyclase, cyclic-AMP phosphodiesterase, protein kinases and phosphoprotein phosphatase in bloodstream forms of Trypanosoma brucei was determined by isopycnic sucrose-gradient centrifugation of post-large-granule extracts. Cyclic-AMP phosphodiesterase was almost entirely soluble whereas adenylate cyclase was membrane-bound. The latter enzyme appeared to be absent from the plasma-membrane fraction but copurified with acid phosphatase and acid phosphodiesterase indicating a possible association with the flagellar pocket. At least two protein kinase activities could be distinguished as based on their distribution profiles in gradients, their preference for exogenously added acceptor protein and their inhibition and stimulation by suramin and nucleoside, respectively. Suramin-sensitive protein kinase co-purified with the plasma-membrane marker alpha-D-glucosidase and a nucleoside-stimulated protein kinase behaved as a typical cell-sap enzyme. Phosphoprotein phosphatase activity was found to be mainly soluble but a small part seemed to be associated with plasma membranes.

Inhibition of NADP-linked malic enzyme from Onchocerca volvulus and Dirofilaria immitis by suramin.

NADP-linked malic enzyme (malate dehydrogenase (oxaloacetate-decarboxylating) NADP+, EC 1.1.1.40) has been partially purified from adult Onchocerca volvulus and Dirofilaria immitis. Suramin was found to inhibit the activity of malic enzyme from both filarial worms. The inhibition constants for suramin were calculated to be 0.011 microM and 0.015 microM for the enzymes from O. volvulus and D. immitis, respectively. In the case of NADP-linked malic enzyme from Trypanosoma brucei and chicken liver the inhibition by suramin was less pronounced. The inhibition constants were found to be 0.8 microM and 2.5 microM for the protozoan and vertebrate enzymes, respectively. The type of inhibition was competitive with respect to malate. The Michaelis constants for malate and pyruvate were determined to be 0.9 and 4.5 mM for O. volvulus and 0.85 and 5.0 mM for D. immitis, respectively. The low Km values for malate compared to those for pyruvate and the about 15-fold greater turnover in the direction of decarboxylation compared to carboxylation indicated that malic enzyme from both filarial sources might be involved in an alternative pathway leading from phosphoenolpyruvate via oxaleacetate, malate and pyruvate to lactate. It is suggested, that the inhibition of malic enzyme activity from O. volvulus by suramin might interfere with the generation of NADPH for biosynthetic reactions.

Hallucinogenic and neuroleptic drug interactions with potential neurotransmitter receptors in parasitic nematodes.

Receptors potentially involved in neurotransmitting have been characterised in the muscle tissue and in whole worms of the nematodes Ascaris suum and Onchocerca volvulus, respectively. Binding studies revealed a high affinity for LSD with apparent KD values of 94 nM for A. suum and 120 nM for O. volvulus, whereas those of the neuroleptics haloperidol, spiperone and mianserin were found to be in the micromolar range. A variety of neurotransmitter antagonists, known to bind with high affinities either to mammalian D1/2 or to 5-HT1/2 receptors, were tested for their ability to bind to the nematode receptor. Results from these displacement experiments using tritiated LSD, mianserin, spiperone and haloperidol show distinct specificities of the nematode receptors compared to known receptor classes of mammals. With respect to this novel specificity, the nematode receptors seem to be unique and clearly distinct from those of the hosts.

Putrescine N-acetyltransferase in Onchocerca volvulus and Ascaris suum, an enzyme which is involved in polyamine degradation and release of N-acetylputrescine.

A novel type of N-acetyltransferase, clearly different from the nuclear and cytosolic polyamine N-acetyltransferases of mammals, was recently found in the intestinal nematode Ascaris suum. The occurrence of this putrescine N-acetylating enzyme has also been noted in the filarial parasite Onchocerca volvulus. The enzyme was partially purified from adults of O. volvulus and A. suum by chromatography on DEAE-cellulose and cadaverine-Sepharose. Substrate specificities of the filarial enzyme resemble those of the N-acetyltransferase from A. suum, with respect to its preference for putrescine and other diamines above polyamines and histones. Additionally, both nematode enzymes acetylated histamine, whereas dopamine and serotonin were not accepted as substrates. The activities of the N-acetyltransferase from O. volvulus and A. suum were potently inhibited by the drug berenil; the type of inhibition was competitive with respect to putrescine. The inhibition constants for berenil were determined as 4.2 and 1.2 microM for the enzymes of O. volvulus and A. suum, the Km values for putrescine were found to be 330 microM and 250 microM, respectively. Putrescine N-acetyltransferase is discussed as a regulatory step in the degradation of excessive polyamines via polyamine oxidase to putrescine. At this branching point, putrescine is retained in the cell for de novo synthesis of spermidine and spermine, catabolized via diamine oxidase or acetylated to a suitable transport product for excretion.

Parasite-specific inhibition of 5'-nucleotidase from Onchocerca volvulus and Dirofilaria immitis by the amoscanate-derivative CGP 8065.

The presence of 5'-nucleotidase was demonstrated in Onchocerca volvulus and Dirofilaria immitis; the bulk of activity was found in the particulate fraction. The enzyme of filarial worms exhibited a broad pH-optimum between 6.4 and 8.0 and substrate specificity for nucleotides compared to glucose-6-phosphate and p-nitrophenyl phosphate. The apparent Km-values for AMP were found to be 0.15 mM and 0.22 mM for the enzyme from O. volvulus and D. immitis, respectively. The activity of 5'-nucleotidase from both filarial worms was effectively inhibited by the filaricidal compound CGP 8065, a dithiocarbamate-derivative of amoscanate, whereas the 5'-nucleotidase from rat liver was not affected. The parasite-specific inhibition by CGP 8065 was found to be reversible and to be competitive with respect to the substrate AMP. The inhibition constants were calculated to be 24 microM and 8 microM for the enzyme from O. volvulus and D. immitis, respectively.

Effect of amoscanate derivative CGP 8065 on aminoacyl-tRNA synthetases in Ascaris suum.

Evidence is provided for the occurrence of a multienzyme complex consisting of several aminoacyl-tRNA synthetases besides 'free enzymes' in Ascaris suum. The molecular mass of this complex was calculated to be about 10(6) daltons, compared to about 150 000 daltons for the seryl-tRNA synthetase. Leucyl-, isoleucyl-, arginyl- and lysyl-tRNA synthestases were found in the high molecular weight fraction. The Michaelis constants of these aminoacyl-tRNA synthetases were found to be in the range of 4 to 10 microM for amino acids and of 0.1 to 1.0 mM for ATP. Leucyl- and isoleucyl-tRNA synthetase interact with the amoscanate-derivative CGP 8065. The inhibition constants were determined to be 34 microM and 8 microM, respectively. The type of inhibition was found to be competitive with respect to ATP. It is proposed that the interference of CGP 8065 with the charging of tRNA might be another target for the chemotherapeutic attack of this amoscanate derivative.

Putrescine-activated S-adenosylmethionine decarboxylase from Acanthamoeba culbertsoni.

Acanthamoeba culbertsoni, the free living pathogenic amoeba responsible for fatal meningoencephalitis, contains an S-adenosylmethionine decarboxylase (EC 4.1.1.50) which is strongly activated by putrescine and to a lesser extent by cadaverine; spermidine, spermine, diaminopropane and 1,6-diaminohexane are inactive. Methylglyoxal bis-(guanylhydrazone) competitively inhibited the enzyme with a Ki value of 123 microM. The enzyme was strongly inhibited by berenil (Ki = 0.5 microM) and to a lesser extent by pentamidine. The putrescine-activated enzyme is inhibited by MgCl2. The apparent molecular weight of 110,000 and its enzymatic properties indicate that the enzyme has characteristics intermediate between the bacterial and eukaryotic S-adenosylmethionine decarboxylases.

Polyamine oxidase from Acanthamoeba culbertsoni specific for N8-acetylspermidine.

Polyamine oxidase plays a key role in the catabolism of polyamines and regeneration of spermidine and putrescine. The mammalian enzyme utilises N1-acetylspermidine, and N8-acetylspermidine, although formed in the mammals, is not catabolised further. We have characterised an enzyme from Acanthamoeba culbertsoni which acts preferentially on N8-acetylspermidine. The highly unstable enzyme was stabilised in the presence of glycerol or dimethylsulphoxide together with spermine and purified 400-fold by a combination of DEAE-cellulose, CM-cellulose, spermine-Sepharose and Sephacryl S-300 chromatography. The enzyme has a pH optimum of 8 and a temperature optimum of 45 degrees C. The relative activities on different substrates are: N8-acetylspermidine 100%, N1-acetylspermine 40%, N1-acetylspermidine 1%, N1,8-diacetylspermidine 1% and N1,12-diacetylspermine 15%. Free polyamines and substrates of monoamine oxidase were not attacked. The enzyme yielded diaminopropane as an end product of catabolism and could be involved in the biosynthesis of this unusual polyamine present in large amounts in this organism.