Nélida S. González

Lack of Arginine Decarboxylase in Trypanosoma cruzi Epimastigotes

Abstract The presence of arginine decarboxylase (ADC) enzymatic activity in Trypanosoma cruzi epimastigotes is still a matter of controversy due to conflicting results published during the last few years. We have investigated whether arginine might indeed be a precursor of putrescine via agmatine in these parasites. We have shown that wild-type T. cruzi epimastigotes cultivated in a medium almost free of polyamines stopped their growth after several repeated passages of cultures in the same medium, and that neither arginine nor ornithine were able to support or reinitiate parasite multiplication. In contrast, normal growth was quickly resumed after adding exogenous putrescine or spermidine. The in vivo labelling of parasites with radioactive arginine showed no conversion of this amino acid into agmatine, and attempts to detect ADC activity measured by the release of CO2 under different conditions in T. cruzi extracts gave negligible values for all strains assayed. The described data clearly indicate that wild-type T. cruzi epimastigotes lack ADC enzymatic activity.

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.

Spermidine is essential for normal proliferation of trypanosomatid protozoa

Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of α‐difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis‐cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.

Anion-exchange chromatography of sugar phosphates with triethylammonium borate

Abstract A method is presented which allows the separation of most of the sugar phosphates on an anion-exchange resin column and permits their almost quantitative recovery. It is based on the use of linear gradient elution by ammonium or triethylammonium tetraborate and on the removal of these salts, after freeze-drying, by distillation with methanol.

Fructokinase from rat liver. I. Purification and properties

Abstract Fructokinase (ATP: d -fructose-1-phosphate transferase, EC 2.7.1.3) from rat liver has been purified 400-fold. The purification procedure involves an acid treatment, a heat step at 65°, (NH4)2SO4 fractionation, chromatography on Sephadex G-100 and finally (NH4)2SO4 extraction. The enzyme appears nearly homogenous by density gradient centrifugation but gives a single peak in sedimentation velocity analysis. Purified liver fructokinase has a K m of 0.46–0.80 mM for fructose and 1.56–1.33 mM for MgATP at a K+ concentration of 0.4 and 0.1 M, respectively. The enzyme also phosphorylates l -sorbose and d -tagatose. No difference could be found in the phosphorylation of the pyranose and furanose forms of fructose. The enzyme is inhibited by p- chloromercuribenzoate and is stable up to 50–55°.

Ornithine decarboxylase from Crithidia fasciculata is metabolically unstable and resistant to polyamine down-regulation

Ornithine decarboxylase (ODC) of Crithidia fasciculata extracts shows maximal activity during exponential growth of the parasite and decreases markedly in the stationary phase. The inhibition of protein synthesis by cycloheximide evoked a rapid loss of enzyme activity with a half‐life of about 30 min. Upon removal of DFMO from Crithidia cultures treated with the drug for 24 h, the ODC activity increased at the same rate as total protein synthesis. The addition of putrescine at high concentrations to parasites cultivated in a synthetic medium showed that Crithidia CDC levels were not reduced by polyamines.

Effect of GTP on the dissociation of 70 S ribosomes.

Zamecnik’s discovery of the GTP requirement for protein synthesis [l] was followed by a series of investigations on the role of this nucleotide both in microbial and mammalian systems. In bacterial extracts GTP is needed in several steps of the protein synthesis process: (a) The binding of N-formyhnethionyl-tRNA to the ribosomes [2,3]. (b) An interaction with the soluble transfer factors T [4,5] and subsequent binding of the aminoacyl-tRNA to the ribosomes [6-lo] . Apparently GTP is not hydrolyzed in these reactions, although there are some conflicting results on this point [6,7,9,10]. (c) The formation of a complex containing ribosomes and transfer factor G [ 1 l] , and the translocation of peptidyl-tRNA [ 121 or N-formylmethionyl-tRNA [ 131 from the aminoacyl (A) to the peptidyl (P) site on the ribosome with concomitant hydrolysis of the nucleotide into GDP and orthophosphate. This communication reports a new effect of GTP: an enhancement of the in vitro dissociation of 70 S ribosomes. The properties and physiological significance of dissociating factors from Bacillus stearothermophilus and Escherichia coli were recently investigated [ 141. The ribosomal dissociation by extracts from E. coli was previously studied by Subramanian et al. [ 151 who did not mention any effect of nucleotides on the reaction.

Studies on dissociation factor of bacterial ribosomes: Effect of antibiotics

Abstract 1. 1. The effect of several antibiotics on the ribosomal dissociation produced in vitro by dissociation factor has been investigated. 2. 2. Sparsomycin, lincomycin, erythromycin, chloramphenicol, fusidic acid and puromycin have little or no effect. The dissociation activity is modified only by antibiotics which interfere with the 30-S subunit functions: the aminoglycosides neomycin B, streptomycin, kanamycin and spectinomycin inhibit the reaction, and the tetracyclines activate it. 3. 3. Neomycin and tetracycline effects occur at the same very low levels which block protein synthesis in vivo . 4. 4. Neomycin, kanamycin and spectinomycin do not abolish the activation by GTP in Bacillus stearothermophilus systems, whereas streptomycin and tetracycline compete with the nucleotide. 5. 5. Some aspects of the ribosomal dissociation mechanism are discussed.

Control of Leishmania mexicana proliferation by modulation of polyamine intracellular levels

Repeated treatments of Leishmania mexicana promastigote cultures with a-difluoromethylornithine could not block proliferation when the parasite was grown in a rich medium. Although the irreversible inhibitor of ornithine decarboxylase was able to abolish the enzymatic activity under these conditions, polyamine depletion was only partial probably due to the uptake of these substances from the external medium. Conversely, when Leishmania was cultivated in a defined medium essentially free of polyamines, a-difluoromethylornithine was able to decrease the growth rate and proliferation was arrested after several passages in the presence of the drug. Parasite multiplication could be resumed by addition of exogenous polyamines, and a strict correlation between Leishmania promastigote growth and intracellular levels of spermidine was observed.

Association of ribosomal subunits: Mechanism of the reaction induced by association factor

The association of ribosomal subparticles into monomers is one of the general properties of bacterial ribosomes that has been more extensively investigated in the last few years. In 1959. TissiZres et al. [ 1 ] found that subunits form 30 S-SO S couples when the Mg’* concentration is increased up to 10 mM or higher. These results have been confirmed by other investigators and many studies on the equilibrium between the monomer and its subunits have now been reported [2-41. This equilibrium can also be shifted towards association by addition of polyamines [2, 5-71, some non-ionic agents such as ethanol and dioxane [8], or aminoglycoside antibiotics, such as neomycin [7, 9-I l] On the other hand ribosomal subparticles tend to associate at relatively low Mg2+ concentrations when they are attached to aminoacylor peptidyl-tRNA [12, 131. Recently we have described a new factor which produces the in vitro association of washed ribosomal subunits (free of aminoacyland peptidyl-tRNA) at 3-5 mM Mg2+ [ 141. This association factor (AF) has been obtained by’extracting ribosomes of Bacillus stearothermophilus with a strong salt solution. AF will not only associate the ribosomal subunits obtained from thermophilic bacteria but also those of Escherichia coli. The splitting of 70 S monomers caused by dissociation factor (DF) can be reversed by the addition of AF [ 141. It has been shown that following dissociation