Papers by Joaquin J B Cannata
Biochemical journal. Cellular aspects, Nov 15, 1979
1. Cell-free extracts from culture epimastigotes of Trypanosoma cruzi contained two forms of NADP... more 1. Cell-free extracts from culture epimastigotes of Trypanosoma cruzi contained two forms of NADP+-linked 'malic' enzyme (EC 1.1. 1.40), I and II, with the same molecular weight but different electrophoretic mobilities and kinetic and regulatory properties. 2. The apparent Km for L-malate was lower for 'malic' enzyme I, with hyperbolic kinetics, whereas the kinetic pattern for 'malic' enzyme II was slightly sigmoidal (h 1.4). The kinetics for NADPH were hyperbolic for 'malic' enzyme I, and very complex for 'malic' enzyme II, suggesting both positive and negative co-operativity. 3. 'Malic' enzyme II was markedly inhibited by adenine nucleotides; AMP was the most effective, at least in the presence of an excess of MnCl2. 'Malic' enzyme I was much less affected by the nucleotides. Both enzyme forms were inhibited by oxaloacetate, competitively towards L-malate, but the apparent K1 for 'malic' enzyme I (9,UM) was 10-fold lower than the value for 'malic' enzyme II. 'Malic' enzyme I1, but not 'malic' enzyme 1, was activated by L-aspartate and succinate (apparent Ka of 0.12 and 0.5mM respectively); the activators caused a decrease in the apparent Km for L-malate and, to a lesser extent, in the apparent Km for NADP+. L-Aspartate, but not succinate, increased the apparent Vmax.. 4. The inhibition by AMP suggests regulation by energy charge, with the L-malate-decarboxylation reaction catalysed by'malic' enzyme II fulfilling a biosynthetic role. The inhibition by oxaloacetate and the activation by succinate are probably involved in the regulation of the 'partial aerobic fermentation' of glucose which yields succinate as final product. The activation by L-aspartate would facilitate the catabolism of this amino acid, when present in excess in the growth medium.
Journal of Biological Chemistry, Aug 1, 1965
![Research paper thumbnail of Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Crithidia fasciculata. Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase](https://attachments.academia-assets.com/107640250/thumbnails/1.jpg)
European journal of biochemistry, Jun 1, 1985
Macromolecules 2,4,6-trimethylheptane.'t5 2,4,6-Trimethyloctane and 2,4,6-trimethylnonane have be... more Macromolecules 2,4,6-trimethylheptane.'t5 2,4,6-Trimethyloctane and 2,4,6-trimethylnonane have been prepared from 2-ethyl-4,6-dimethylheptanoic acid methyl ester and 2-propyl-4,6-dimethyl heptanoic acid methyl ester after: (1) reduction to alcohol with LiAlH4 in ethyl ether, (2) chlorination of the alcohol with dichlorotri-phenylph~sphorane,'~ and (3) transformation of the halomethyl group into the lithiomethyl group in diethyl ether and subsequent hydrolysis with HzO. 2-Ethyl-4,6-dimethylheptanoic acid and 2-propyl-4,6-dimethylheptanoic acid have been prepared after reaction of 1bromo-2,4-dimethylpentane with diethylethylsodiomalonate and diethylpropylsodiomalonate respectively and subsequent hydrolysis and decarboxylation."J' Proton noise decoupled I3C NMR spectra were measured a t 140 "C in 1,2,4-trichlorobenzene solutions (10% v/v) by adding 1% hexamethyldisiloxane (HMD) as internal reference. These conditions where chosen since they are typical for the analysis of hydrocarbon polymers. An HX-90 Bruker spectrometer operating a t 22.63 MHz in the PFT mode was used as described p r e v i~u s l y .~
![Research paper thumbnail of Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Trypanosoma cruzi. Evidence of the presence of two alanine pools and of two CO2 fixation reactions](https://attachments.academia-assets.com/107640248/thumbnails/1.jpg)
European journal of biochemistry, Sep 1, 1990
Macromolecules 2,4,6-trimethylheptane.'t5 2,4,6-Trimethyloctane and 2,4,6-trimethylnonane have be... more Macromolecules 2,4,6-trimethylheptane.'t5 2,4,6-Trimethyloctane and 2,4,6-trimethylnonane have been prepared from 2-ethyl-4,6-dimethylheptanoic acid methyl ester and 2-propyl-4,6-dimethyl heptanoic acid methyl ester after: (1) reduction to alcohol with LiAlH4 in ethyl ether, (2) chlorination of the alcohol with dichlorotri-phenylph~sphorane,'~ and (3) transformation of the halomethyl group into the lithiomethyl group in diethyl ether and subsequent hydrolysis with HzO. 2-Ethyl-4,6-dimethylheptanoic acid and 2-propyl-4,6-dimethylheptanoic acid have been prepared after reaction of 1bromo-2,4-dimethylpentane with diethylethylsodiomalonate and diethylpropylsodiomalonate respectively and subsequent hydrolysis and decarboxylation."J' Proton noise decoupled I3C NMR spectra were measured a t 140 "C in 1,2,4-trichlorobenzene solutions (10% v/v) by adding 1% hexamethyldisiloxane (HMD) as internal reference. These conditions where chosen since they are typical for the analysis of hydrocarbon polymers. An HX-90 Bruker spectrometer operating a t 22.63 MHz in the PFT mode was used as described p r e v i~u s l y .~
Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. Th... more Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.
Molecular and Biochemical Parasitology, 1999
ABSTRACT Three molecular forms of serine hydroxymethyltransferase (SHMT) have been detected in ch... more ABSTRACT Three molecular forms of serine hydroxymethyltransferase (SHMT) have been detected in choanomastigotes of Crithidia fasciculata by DEAE-cellulose chromatography. The three isoforms (named SHMT I, II, and III) presented small differences in charge and molecular weight. Digitonin treatment of intact cells suggested that SHMT III is cytosolic, whereas the other two isoforms are particle bound, one being mitochondrial (SHMT I) and the other one very likely glycosomal (SHMT II). The three SHMT isoforms were purified to homogeneity, and their physicochemical and kinetic properties studied. Determination of their native and subunit molecular masses revealed that all of them have a tetrameric structure. The three isoforms were shown to be PLP-dependent enzymes after L-cysteine and hydroxylamine hydrochloride treatments. They showed similar pH optima, bimodal kinetics for L-serine and Michaelis-Menten kinetics for THF.
Molecular and Biochemical Parasitology, Jun 1, 2004
Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. Th... more Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.
Parasitology, Jan 5, 2018
Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the a... more Several ortho-naphthoquinones (o-NQs) have trypanocidal activity against Trypanosoma cruzi, the aetiological agent of Chagas disease. Previously, we demonstrated that the aldo-keto reductase from this parasite (TcAKR) reduces o-NQs, such as β-lapachone (β-Lap) and 9,10-phenanthrenequinone (9,10-PQ), with concomitant reactive oxygen species (ROS) production. Recent characterization of TcAKR activity and expression in two T. cruzi strains, CL Brener and Nicaragua, showed that TcAKR expression is 2.2-fold higher in CL Brener than in Nicaragua. Here, we studied the trypanocidal effect and induction of several death phenotypes by β-Lap and 9,10-PQ in epimastigotes of these two strains. The CL Brener strain was more resistant to both o-NQs than Nicaragua, indicating that greater TcAKR activity is unlikely to be a major influence on o-NQ toxicity. Evaluation of changes in ROS production, mitochondrial membrane potential, phosphatidylserine exposure and monodansylcadaverine labelling eviden...
Biochemical Journal, 1989
Phosphoenolpyruvate carboxykinase (PEPCK) was purified to homogeneity from the moderately halophi... more Phosphoenolpyruvate carboxykinase (PEPCK) was purified to homogeneity from the moderately halophilic bacterium Vibrio costicola. The enzyme is monomeric, with an Mr of 62,000, as determined by the Svedberg equation, by using values of s0(20,w) 4.4 x 10(-13) s, D20,w 6.13 x 10(-7) cm2.s-1 and v 0.719 cm3.g-1. Compared with other, non-halophilic, PEPCKs, the enzyme from V. costicola had a significantly lower total content of hydrophobic amino acids. The contents of glycine and serine were higher in the V. costicola enzyme (16.7 and 10.22% respectively) than in the non-halophilic PEPCKs (6.8-9.6% and 4.67-6.28% respectively). These results resemble those obtained by De Médicis & Rossignol [(1979) Experientia 35, 1546-1547] with the pyruvate kinase from V. costicola, and agree with the proposal by Lanyi [(1974) Bacteriol. Rev. 38, 272-290] of partial replacement of hydrophobic amino acids by glycine and serine to maintain the balance between hydrophobic and hydrophilic forces in halophi...
Biochemistry and molecular biology international, 1995
The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetat... more The phosphoenolpyruvate carboxykinase (PEPCK) from Vibrio costicola catalyzed a 14CO2-oxaloacetate exchange reaction with an unusual nucleotide specificity. ATP gave the higher apparent catalytic efficiency (Vmax/Km, 6.78), followed by GTP (1.30), CTP (0.87) and ITP (0.66). Maximal activity required a divalent cation; CdCl2 and MgCl2 synergistically activated the enzyme, when added in the presence of MnCl2. The sigmoidal saturation curve for MnCl2 (apparent n 2.11) was converted into a hyperbola by 0.01 mM CdCl2 (apparent n 1). The results suggest a double role of the divalent cation in the reaction mechanism, namely as part of the MeATP2- substrate and as free Me2+. Mn2+ would be the best for the first, and Cd2+ for the second role. Preincubation with 0.01 mM CdCl2 increased the activity of the enzyme assayed with MgATP2- through an increase in Vmax; addition of CdCl2 to the reaction mixture elicited further activation, through a 17-fold decrease in the apparent Km for MgATP2-. The...
Biochimica et Biophysica Acta, 1959
Antimicrobial agents and chemotherapy, May 8, 2016
Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Tr... more Benznidazole (Bz), the drug used for treatment of Chagas' disease (caused by the protozoan Trypanosoma cruzi), is activated by a parasitic NADH-dependent nitroreductase (NTR I). However, several studies have shown that other enzymes are involved. The aim of this study was to evaluate whether the aldo-keto reductase from T. cruzi (TcAKR), a NADPH-dependent oxido-reductase previously described by our group, uses Bz as substrate. We demonstrated that both recombinant and native TcAKR reduce Bz by using NADPH, but not NADH, as co-factor. TcAKR-overexpressing epimastigotes showed higher NADPH-dependent Bz reductase activity and a 50% inhibitory concentration (IC50) value for Bz 1.8-fold higher than controls, suggesting that TcAKR is involved in Bz detoxification instead of activation. To understand the role of TcAKR in Bz metabolism, we studied TcAKR expression and NADPH/NADH-dependent Bz reductase activities in two T. cruzi strains with differential susceptibility to Bz: CL and Nica...
The causative agent of Chagas disease, Trypanosoma cruzi, metabolizes glucose through two major p... more The causative agent of Chagas disease, Trypanosoma cruzi, metabolizes glucose through two major pathways: glycolysis and the pentose phosphate pathway. Glucose is taken up via one facilitated transporter and its catabolism by the glycolytic pathway leads to the excretion of reduced products, succinate and l‑alanine, even in the presence of oxygen; the first six enzymes are located in a peroxisome‑like organelle, the glycosome, and the lack of regulatory controls in hexokinase and phosphofructokinase results in the lack of the Pasteur effect. All of the enzymes of the pentose phosphate pathway are present in the four major stages of the parasite’s life cycle, and some of them are possible targets for chemotherapy. The gluconeogenic enzymes phosphoenolpyruvate carboxykinase and fructose‑1,6‑bisphosphatase are present, but there is no reserve polysaccharide.
Molecular and Biochemical Parasitology, 1987
Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28 degrees C, reach a station... more Axenic culture amastigote-like forms of Trypanosoma cruzi, grown at 28 degrees C, reach a stationary phase after two generations, and differentiate to epimastigotes, which then resume growth. Axenic culture amastigotes readily ferment glucose to succinate and acetate, and do not excrete NH3; they have high activities of hexokinase and phosphoenolpyruvate carboxykinase, and very low citrate synthase activity; cytochrome o is absent, and cytochrome b-like is present at a very low level. Epimastigotes catabolize glucose and produce succinate and acetate at a considerably lower rate; they exhibit lower levels of hexokinase and carboxykinase, and much higher levels of citrate synthase and cytochromes o and b-like. They catabolize amino acids, as shown by excretion of NH3 to the medium. The results suggest that axenic culture amastigotes have an essentially glycolytic metabolism, and they acquire the ability to oxidize substrates such as amino acids only after differentiation to epimastigotes.
Mol Biochem Parasitol, 1995
Phosphoenolpyruvate carboxykinase (PEPCK) has been purified to homogeneity from epimastigotes of ... more Phosphoenolpyruvate carboxykinase (PEPCK) has been purified to homogeneity from epimastigotes of the Tul 0 strain of Trypanosoma cruzi. The physicochemical parameters determined allowed the calculation of an average molecular mass of 120 kDa; the subunit molecular mass, about 61 kDa, is in good agreement with the value of 58.6 kDa recently determined from the sequence by Sommer et al. (FEBS Lett. 359 (1994) 125-129). The PEPCK from T. cruzi presented, in addition to its molecular mass, typical properties of other ATP-linked PEPCKs, namely strict specificity for ADP in the carboxylation reaction and lower specificity in the decarboxylation and exchange reactions, and synergistic activation by CdCl, or MgCl, when added in addition to MnCl,. The enzyme presented hysteretic behaviour, shown by a lag period in the carboxylation reaction, which was affected by dilution and preincubation. The decarboxylation reaction catalyzed by the T. cruzi PEPCK was not inhibited by excess of ATP-Mn. The apparent K, values for the carboxylation reaction, including the low value for PEP (0.035 mM) are compatible with an important role of PEPCK, as suggested by previous NMR experiments, on the CO, fixation in vivo which leads to succinate excretion during aerobic fermentation of glucose.
Molecular and Biochemical Parasitology, Oct 1, 2010
The Journal of biological chemistry, 1963
The Biochemical journal, 2001
We found that, as in African trypanosomes, endogenous phospholipase A(1) (Plase A(1)) activity ca... more We found that, as in African trypanosomes, endogenous phospholipase A(1) (Plase A(1)) activity can catalyse extensive deacylation of phospholipids upon cell death in all life stages of Trypanosoma cruzi. A major lysosomal Plase A(1) was purified and characterized. The enzyme products can explain the lesions surrounding degenerating T. cruzi cells in host tissues. Thus Plase A(1) emerges as a target to block pathogenesis in trypanosomal infections.
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Papers by Joaquin J B Cannata