Arginine kinase: a common feature for management of energy reserves in African and American flagellated trypanosomatids

This work reports the characterization of an arginine kinase in the unicellular parasitic flagellate Trypanosoma brucei, the etiological agent of human sleeping sickness and Nagana in livestock. The arginine kinase activity, detected in the soluble fraction obtained from procyclic forms, had a specific activity similar to that observed in Trypanosoma cruzi, about 0.2 micromol min(-1) mg(-1). Western blot analysis of T. brucei extracts revealed two bands of 40 and 45 kDa. The putative gene sequence of this enzyme had an open reading frame for a 356-amino acid polypeptide, one less than the equivalent enzyme of T. cruzi. The deduced amino acid sequence has an 82% identity with the arginine kinase of T. cruzi, and highest amino acid identities of both trypanosomatids sequences, about 70%, were with arginine kinases from the phylum Arthropoda. In addition, the amino acid sequence possesses the five arginine residues critical for interaction with ATP as well as two glutamic acids and one cysteine required for arginine binding. The finding in trypanosomatids of a new phosphagen biosynthetic pathway, which is not present in mammalian host tissues, suggests this enzyme as a possible target for chemotherapy.     

Kinetics of hydrolysis of phenylthiazolones of arginine, homoarginine, norarginine, and canaavanine by trypsin.

Phenylthiazolones (PTAs) of arginine and its homologs and analogs, homoarginine, norarginine (alpha-amino-gamma-guanidinobutyric acid), canavanine, and gamma-hydroxyarginine, were prepared. A steady-state kinetic analysis of the trypsin [EC 3.4.21.4]-catalyzed hydrolysis reactions was carried out and the kinetic parameters for these internal thioesters were compared with those for normal linear ester substrates. PTA-gamma-hydroxyarginine was so labile that hydrolysis by the enzyme could not be followed. PTA-arginine has a specificity constant (Kcat/Km) comparable to that for the Nalpha-unblocked arginine ester substrate, though the value is about 0.1% of that for a specific ester substrate, Nalpha-tosylarginine methyl ester. PTA derivatives of canavanine and homoarginine were hydrolyzed with Kcat/Km walues of the same order of magnitude as that for PTA-arginine. However, PTA-noraginine was much less susceptible to tryptic hydrolysis that PTA-homoarginine, while the linear esters of norarginine are known to be more susceptible than those of homoarginine.     

Biochemical and immunological studies of protein kinase C from Trypanosoma cruzi.

Phorbol ester binding was studied in protein kinase C-containing extracts obtained from Trypanosoma cruzi epimastigote forms. Specific 12-O-tetradecanoyl phorbol 13-acetate, [3H]PMA, or 12,13-O-dibutyryl phorbol, [3H]PDBu, binding activities, determined in T. cruzi epimastigote membranes, were dependent on ester concentration with a Kd of 9x10(-8) M and 11.3x10(-8) M, respectively. The soluble form of T. cruzi protein kinase C was purified through DEAE-cellulose chromatography. Both protein kinase C and phorbol ester binding activities co-eluted in a single peak. The DEAE-cellulose fraction was further purified into three subtypes by hydroxylapatite chromatography. These kinase activity peaks were dependent on Ca2+ and phospholipids and eluted at 40 mM (PKC I), 90 mM (PKC II) and 150 mM (PKC III) phosphate buffer, respectively. Western blot analysis of the DEAE-cellulose fractions, using antibodies against different isoforms of mammalian protein kinase C enzymes, revealed that the parasite expresses high levels of the alpha-PKC isoform. Immunoaffinity purified T. cruzi protein kinase C, isolated with an anti-protein kinase C antibody-sepharose column, were subjected to phosphorylation in the absence of exogenous phosphate acceptor. A phosphorylated 80 kDa band was observed in the presence of Ca2+, phosphatidylserine and diacylglycerol.     

Arginine Transport in the Culture Form of Trypanosoma cruzi

SYNOPSIS. Arginine in Trypanosoma cruzi is taken up by a mediated transport system; a large non-saturable component is also involved. Arginine transport specificity in T. cruzi is unusual since lysine and histidine do not inhibit its uptake. Glycine and valine are partially competitive inhibitors and homoarginine, alanine and methionine are completely competitive. At least one arginine transport site is so specific that homoarginine will not react with it. Apparently the diamino acid transport systems of T. cruzi have undergone major evolutionary modifications.     

An expanded adenylate kinase gene family in the protozoan parasite Trypanosoma cruzi

Adenylate kinases supply energy routes in cellular energetic homeostasis. In this work, we identified and characterized the adenylate kinase activity in extracts from the flagellated parasite Trypanosoma cruzi, the causative agent of Chagas' disease. Adenylate kinase activity was detected in different subcellular fractions and the cytosolic isoform was biochemically characterized. Cytosolic adenylate kinase specific activity increases continuously during the epimastigote growth and is down-regulated when other soluble phosphotransferase, arginine kinase, is overexpressed. Six different genes of adenylate kinase isoforms were identified and the mRNA expression was confirmed by RT-PCR and Northern Blot. Three open reading frames coding for different enzyme isoforms named TzADK1, TzADK2 and TzADK5 were cloned and functionally expressed in E. coli. This work reports an unusually large number of genes of adenylate kinases and suggests a coordinated regulation of phosphotransferase-mediated ATP regenerating pathways in the unicellular parasite Trypanosoma cruzi.     

Stimulation of proteinase biosynthesis by canavanine in streptococcus faecalis var. liquefaciens

l-Canavanine, an analogue of arginine, was found to stimulate the synthesis of an extracellular proteinase in Streptococcus faecalis var. liquefaciens. Cells grown in a synthetic medium containing 10(-4)m arginine and 10(-4)m canavanine produced almost twice as much proteinase as cells grown in 2 x 10(-3)m arginine alone; total growth was the same in both media. Hydrolyzed proteinase samples were analyzed for arginine and canavanine by means of paper chromatography and electrophoresis. Arginine, but not canavanine, was detected in the purified enzyme sample.     

Arginine metabolism in Trypanosoma cruzi is coupled to parasite stage and replication

L-Arginine plays an essential role in the energetic metabolism of Trypanosoma cruzi. In this work we propose a relationship between L-arginine uptake, arginine kinase activity and the parasite replication ability. In epimastigote cultures L-arginine uptake decreases continuously accompanying a cell replication rate reduction. The use of conditioned or fresh medium mimics uptake variations. Interestingly, in non-replicative trypomastigote cells, L-arginine uptake was undetectable. The association between L-arginine uptake and cell replication was demonstrated using the antimitotic agent hydroxyurea. Arginine kinase, the enzyme responsible for phosphoarginine and ATP synthesis, also shows a differential activity in epimastigote and trypomastigote parasite stages.     

The Effect of Some Protein and Non-Protein Amino Acids on the Growth of Cladosporium herbarum and Trichothecium roseum

The effect of ten amino acids as the sole nitrogen source for the growth of Cladosporium herbarum (Link.) Fr. and Trichothecium roseum (Bull.) Link. was studied in order to clarify the fungus-host plant relationship. Special attention was paid to some rare non-protein amino acids of legumes. The best nitrogen sources for both fungi were γ-aminobutyric acid, arginine, serine and proline. Cladosporium could use homoarginine and canavanine, but these two amino acids were not used by Trichothecium when each was given as the only nitrogen source. Both fungi utilized ornithine, homoserine and a,γ-diaminobutyric acid to a limited extent. Pipecolic acid was not growth promoting. The growth-retarding effects of rare non-protein amino acids (homoarginine, canavanine, a,γ-diaminobutyric acid and pipecolic acid) were usually reversed by higher concentrations of their normal analogues. It is possible that rare non-protein amino acids may slightly protect the host plant against fungal infections, but there are clear differences between fungi in their reaction to non-protein amino acids.     

Effects of protein kinase and phosphatidylinositol-3 kinase inhibitors on growth and ultrastructure of Trypanosoma cruzi

An increasing number of protein kinases (PKs) of parasitic protozoa are being evaluated as drug targets. Some PK inhibitors display antiproliferative effects on protozoa. We tested three PK inhibitors on the growth and ultrastructure of epimastigotes of Trypanosoma cruzi and the effect of these drugs on intracellular amastigotes. They were staurosporine (serine/threonine kinase inhibitor), genistein (tyrosine kinase inhibitor), and wortmannin (phosphatidylinositol 3' (PI3) kinase inhibitor). All drugs inhibited epimastigote growth at the concentrations tested. Wortmannin inhibited parasite growth at the lowest concentrations. However, staurosporine was the most effective after 24 h treatment and genistein caused the stronger inhibition during the whole treatment (60-70% inhibition). The IC50 were: staurosporine: 6.43+/-1.28 microM; genistein: 6.54+/-1.86 microM; and wortmannin: 0.056+/-0.014 microM. These PK inhibitors had strong ultrastructural effects on the epimastigotes: abnormal chromatin condensation of the nucleus; loose flagellar membrane with the formation of blebs; incomplete cell division; autophagosomes and myelin-like figures. These drugs did not interfere with the division of intracellular amastigotes or with its differentiation to trypomastigotes. However, as trypanosomes have kinomes that contain a large set of protein kinases and phosphatases, PKs should not be disregarded as an important target for chemotherapy of Chagas disease.     

Trypanosoma cruzi: Oxidative stress induces arginine kinase expression

Trypanosoma cruzi arginine kinase is a key enzyme in cell energy management and is also involved in pH and nutritional stress response mechanisms. T. cruzi epimastigotes treated with hydrogen peroxide presented a time-dependent increase in arginine kinase expression, up to 10-fold, when compared with untreated parasites. Among other oxidative stress-generating compounds tested, only nifurtimox produced more than 2-fold increase in arginine kinase expression. Moreover, parasites overexpressing arginine kinase showed significantly increased survival capability during hydrogen peroxide exposure. These findings suggest the participation of arginine kinase in oxidative stress response systems.     

L-arginine uptake and L-phosphoarginine synthesis in Trypanosoma cruzi

A very specific L-arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L-arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1-57.5 pmol.min-1 per 3 x 10(7) cells and between 4.2-5.5 microM, respectively. The calculated activation energy and Q10 were 31.1 KJ.mol-1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L-arginine. Cells retained the labeled amino acid independently of the presence or absence of exogenous L-arginine. The specificity of L-arginine uptake was demonstrated by competition assays in the presence of 80-fold molar excess of natural amino acids and several L-arginine derivatives. The highest levels of inhibition were caused by L-homoarginine, D-arginine, L-canavanine, L-ornithine, and L-citrulline. L-arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5-8.5. The major product of L-arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.     

Differential behaviour of glucose 6-phosphate dehydrogenase in two morphological forms of Trypanosoma cruzi

1. Glucose 6-phosphate dehydrogenase activity (EC 1.1.1.49) of two morphological forms of Trypanosoma cruzi, epimastigotes and metacyclics, are reported. 2. The kinetic behaviour and some of the kinetic parameters of the enzyme in both forms were studied. The enzymes showed a simple Michaelis-Menten kinetic. 3. The activity in epimastigote forms was alway higher than the metacyclic ones. At subsaturating concentrations of substrate was almost 10-fold higher, whereas at saturating concentrations was about 2-fold higher. 4. In epimastigote forms the specific activity and Km values, at pH 7.5 and 37 degrees C, was found to be 142 mUnits x mg-1 of protein and 0.23 mM, respectively. 5. In the same conditions, the specific activity and Km values in metacyclic forms was 75 mUnits x mg-1 of protein and 1.06 mM, respectively. 6. A possible role in the carbohydrate metabolism of glucose 6-phosphate dehydrogenase in both forms of Trypanosoma cruzi is discussed.     

Studies of L-canavanine incorporation into insectan lysozyme.

L-Canavanine is incorporated into the lysozyme synthesized, in response to administration of bacterial cell wall materials, by canavanine-treated larvae of the tobacco hornworm Manduca sexta (Sphingidae). Maximum canavanine incorporation into M. sexta lysozyme occurs when the larvae are provided 1 mg of canavanine g-1 fresh body weight. Analysis of canavanine-containing lysozyme purified from these insects reveals that 21% of the arginine residues are replaced by canavanine; this residue substitution results in a loss of 49.5% of the catalytic activity. When the larvae are provided 0.5 mg of canavanine g-1, 16.5% of the arginine residues are substituted by canavanine and 39.5% of the catalytic activity is lost. Canavanine is also incorporated into the lysozyme induced by canavanine-treated pupae of the giant silk moth Hyalophora cecropia (Saturnidae). In contrast, replacement of 17% of the arginine in H. cecropia lysozyme by canavanine fails to affect the catalytic activity. We have determined the primary structure of M. sexta lysozyme and compared it with the primary structure of H. cecropia lysozyme which has been described elsewhere. M. sexta lysozyme has an arginine at positions 23, 42, and 107. H. cecropia contains serine, lysine, and lysine, respectively, at these locations. The ability of incorporated canavanine to inhibit M. sexta lysozyme activity selectively may result from the fact that replacement of any one of the 3 arginine residues at position 23, 42, or 107 by canavanine causes the loss of catalytic activity.     

Protein kinase CK1 from Trypanosoma cruzi

A protein kinase activity, which uses casein as a substrate, has been purified to homogeneity from the epimastigote stage of Trypanosoma cruzi, by sequential chromatography on Q sepharose, heparin sepharose, phenyl sepharose, and alpha-casein agarose. An apparent molecular weight of 36,000 was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration chromatography and sedimentation analyses demonstrated that the purified native enzyme is a monomer with a sedimentation coefficient of 2.9 S. The hydrodynamic parameters indicated that the shape of the protein is globular with a frictional ratio f/f(o) = 1.36 and a Stokes radius of 27.7 A. When two selective peptide substrates for protein kinases CK1 and CK2 were used (RRKDLHDDEEDEAM. SITA and RRRADDSDDDDD, respectively), the purified kinase was shown to predominantly phosphorylate the CK1-specific peptide. Additionally, the enzyme was inhibited by N-(2-amino-ethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inactivator of CK1s from mammals. Based on these results, we concluded that the purified kinase corresponds to a parasite CK1.     

Molecular cloning and expression of the catalytic subunit of protein kinase A from Trypanosoma cruzi

The activation of protein kinase A (cyclic adenosine monophosphate-dependent protein kinase) by cyclic adenosine monophosphate is believed to play an important role in regulating the growth and differentiation of Trypanosoma cruzi. A PCR using degenerate oligonucleotide primers against conserved motifs in the VIb and VIII subdomains of the ACG family of serine/threonine protein kinases was utilised to amplify regions corresponding to the parasite homologue of the protein kinase A catalytic subunit. This putative protein kinase A fragment was used to isolate the entire gene from T. cruzi genomic libraries. The deduced 329 amino acid sequence of this gene contained all of the signature motifs of known protein kinase A catalytic subunit proteins. The recombinant protein expressed in Escherichia coli was shown to phosphorylate Kemptide, a synthetic peptide substrate of protein kinase A, in a protein kinase inhibitor (PKI)-inhibitory manner. Immunoprecipitation with polyclonal antisera raised against recombinant protein of this gene was able to pull-down PKI-inhibitory phosphotransferase activity from epimastigote lysates. Immunoblot and Northern blot analyses, in combination with enzyme activity assays, revealed that this gene was a stage-regulated enzyme in T. cruzi with higher levels and activity being present in epimastigotes compared with amastigotes or trypomastigotes. Overall these studies indicate that the cloned gene encodes an authentic protein kinase A catalytic subunit from T. cruzi and are the first demonstration of PKI-inhibitory phosphotransferase activity in an expressed protozoan protein kinase A catalytic subunit.     

3-[2-Amino-2-imidazolin-4-yl] alanine, 2-[2-amino-2-imidazolin-4-yl] acetic acid, 2-aminoimidazole and other guanidine derivatives in the tephrosieae

The distribution of enduracididine, 2-[2-amino-2-imidazolin-4-yl] acetic acid, 2-aminoimidazole, canavanine, homoarginine, γ-hydroxyhomoarginine and other unidentified guanidino compounds in seeds of spp. of the Tephrosieae is described. Within Lonchocarpus enduracididine and 2-[2-amino-2-imidazolin-4-yl] acetic acid were found only in American spp. and canavanine only in African spp.     

Alterations of the microcirculatory network during the clearance of epimastigote forms of Trypanosoma cruzi: an intravital microscopic study

The distribution of epimastigote forms of Trypanosoma cruzi in the microcirculatory network and the vessel alterations were observed using an intravital microscopy technique. Immediately after intravenous inoculation of 2 x 10(6) epimastigote suspension into normal mice, parasites were seen as circulating clumps, and their retention at some sites of the endothelium of venules and capillaries was observed. Injection of 2 x 10(7) and 2 x 10(8) parasite suspensions induced, respectively, intermittent or total stasis of venules and capillaries, probably via obstruction by clumping. The mobility of epimastigotes in the clumps indicates that parasites were alive in the lumen of vessels. The retention of clumps in the capillaries, although intense, could only be observed when labeled parasites were inoculated. These results suggest that the rapid clearance of epimastigote forms of T. cruzi from the blood circulation of mice may be due to the retention of parasites to the endothelium of venules and capillaries that, in turn, may facilitate phagocytosis. This may be a mechanism by which mice are able to eliminate epimastigote forms from the circulation. These findings are consistent with our previous observations showing that epimastigotes are not lysed by complement activation but are phagocytosed and destroyed by a distinct population of blood cells.     

Parallel regulation of arginine transport and nitric oxide synthesis by angiotensin II in vascular smooth muscle cells role of protein kinase C

Summary Experiments were performed to characterize arginine transport in vascular smooth muscle cells (SMCs) and the effect of angiotensin II (Ang II) on this process. In addition, the role of arginine transport in the cytokineinduced nitric oxide (NO) production was assessed. Arginine transport takes place through Na⁺-independent (60%) and Na⁺-dependent pathways (40%). The Na⁺-independent arginine uptake appears to be mediated by system y⁺ because of its sensitivity to cationic amino acids such as lysine, ornithine and homoarginine. The transport system was relatively insensitive to acidification of the extracellular medium. By contrast, the Na⁺-dependent pathway is consistent with system B^0,+ since it was inhibited by both cationic and neutral amino acids (i.e., glutamine, phenylalanine, and asparagine), and did not accept Li⁺ as a Na⁺ replacement. Treatment of SMCs with 100nM Ang II significantly inhibited the Na⁺-dependent arginine transport without affecting systems y⁺, A, and L. This effect occurred in a dose-dependent manner (IC₅₀ of 8.9 ± 0.9nM) and is mediated by the AT-1 receptor subtype because it was blocked by DUP 753, a non-peptide antagonist of this receptor. The inhibition of system B^0,+ by Ang II is mediated by protein kinase C (PKC) because it was mimicked by phorbol esters (phorbol 12-myristate 13-acetate) and was inhibited by staurosporine. Ang II also inhibited the IL-1 induced nitrite accumulation by SMCs. This action was also inhibited by staurosporine and reproduced with phorbol esters, suggesting a coupling between arginine uptake and NO synthesis through a PKC-dependent mechanism. However, arginine supplementation in the medium (10mM) failed to prevent the inhibitory action of Ang II on NO synthesis. These findings suggest that although Ang II inhibits concomitantly arginine transport and NO synthesis in SMCs, the reduction of NO synthesis is not associated with alterations in the cellular transport of arginine.Abbreviations Arg arginine - Orn ornithine - HmR homoarginine - Lys lysine - Gln glutamine - Asn asparagine - His histidine - Phe phenylalanine - Leu leucine - Cys Cysteine - Ala alanine - Ser serine - Thr threonine - Glu glutamate - mAIB -methyl-aminoisobutyric acid - BCH bicycloaminoheptane     

Surface charge of trypanosoma cruzi. Binding of cationized ferritin and measurement of cellular electrophoretic mobility.

The surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi was evaluated by means of binding of cationized ferritin to the cell surface as visualized by electron microscopy, and by direct measurements of the cellular microelectrophoretic mobility (EPM). Epimastigote forms had a mean EPM of -0.52 micrometer-s-1-V-1-cm and were lightly labeled with cationized ferritin. In contrast, bloodstream trypomastigotes had a much higher EPM (-1.14), and the surface was heavily labeled with cationized ferritin. When trypomastigotes from staionary phase cultures were isolated on DEAE cellulose columns, the mean EPM was found to be significantly lower (-0.63), and labeling with cationized ferritin decreased. With a mixed population containing epimastigote, trypomastigote, and intermediate forms, EPM values ranging between -0.70 to -1.14 were found. From these observations we conclude that there is a definite increase in negative surface charge during development from epi- to trypomastigote forms of T. cruzi.