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J J Cazzulo

Publications and source records attributed to J J Cazzulo.

At least 91 records · Page 5Linked to original sources
PubMed1987

Aerobic glucose fermentation by Trypanosoma cruzi axenic culture amastigote-like forms during growth and differentiation to epimastigotes.

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.

Amino Acids↗
PubMed1987

Characterization by electrophoretic zymograms of 19 Trypanosoma cruzi clones derived from two chronic chagasic patients.

1. Electrophoretic patterns of aspartate aminotransferase, glucose-6-phosphate dehydrogenase, phosphoglucomutase, glucose-phosphate isomerase, malic enzyme and alcohol dehydrogenase have been analyzed in extracts from Trypanosoma cruzi, Tulahuén strain, 19 clones derived from isolates obtained from two chronic chagasic patients from Argentina and from Brazilian stocks Silvio X10/1 (zymodeme 1), Esmeraldo/1 (zymodeme 2), and CAN-III/1 (zymodeme 3). 2. The clones isolated from one of the patients were genetically heterogeneous. 3. Phosphoglucomutase and glucose phosphate isomerase patterns for the clones analyzed clearly differ from those of the Brazilian stocks. 4. Grouping of clones on the basis of isozyme patterns showed some correlation with that based on total DNA per organism. 5. Under the experimental conditions used, the polyacrylamide gel electrophoresis micromethod employed was advantageous over starch gel electrophoresis.

Alcohol Dehydrogenase↗
PubMed1987

Inhibition of growth and respiration of Leishmania mexicana by the antitumor agent lonidamine.

1. The antitumor drug lonidamine inhibited growth of promastigotes of Leishmania mexicana in axenic culture. 2. Fifty percent inhibition was attained at 0.42 mM, and was reflected mainly in an increase in lag time, with less effect on final cell yield. 3. The drug was leishmanistatic, since when a non-growing culture in the presence of 0.5 mM lonidamine was centrifuged and the cells resuspended in fresh medium, growth started and reached the control value. 4. Both coupled and FCCP-uncoupled respiration of intact promastigotes were inhibited by lonidamine; 50% inhibition was attained at 0.5 and 0.4 mM, respectively. 5. The results suggested that the mechanism of inhibition of growth of L. mexicana is, as proposed in the case of Trypanosoma cruzi epimastigotes and Trypanosoma brucei procyclic trypomastigotes, through inhibition of the energy metabolism.

Animals↗
PubMed1985

Carbon-13 nuclear magnetic resonance analysis of [1-13C]glucose metabolism in Crithidia fasciculata. Evidence of CO2 fixation by phosphoenolpyruvate carboxykinase.

The non-invasive technique of 13C nuclear magnetic resonance was applied to study glucose metabolism in vivo in the insect parasite Crithidia fasciculata. It was found that under anaerobic conditions [1-13C]glucose underwent a glycolytic pathway whose main metabolic products were identified as [2-13C]ethanol, [2-13C]succinate and [1,3-13C2]glycerol. These metabolites were excreted by C. fasciculata into the incubation medium, while in the cells [3-13C]phosphoenolpyruvate was also detected in addition to the aforementioned compounds. The C3 acid is apparently the acceptor of the primary CO2 fixation reaction, which leads in Trypanosomatids to the synthesis of succinate. By addition of sodium bicarbonate to the incubation mixture L-[3-13C]malate was detected among the excretion products, while the ethanol:succinate ratio of 2.0 in the absence of bicarbonate changed to a ratio of 0.6 in the presence of the latter. This was due to a shift of the balance between carboxylation of phosphoenolpyruvate, leading to succinate, and pyruvate decarboxylation leading to ethanol. The addition of 25% 2H2O to the incubation mixture led to the formation of [2-13C, 2-2H]ethanol derived from the prior incorporation of 2H+ into pyruvate in the reactions mediated by either pyruvate kinase or malic enzyme. However, no 2H+ incorporation into L-malate was detected, excluding the possibility that the latter was formed by carboxylation of pyruvate, and lending support to the idea that L-malate results from the carboxylation of phosphoenolpyruvate to oxaloacetate by phosphoenolpyruvate carboxykinase. The formation of [2-13C, 2-2H]-succinate under the same conditions reflected the uptake of 2H+ during the reduction of fumarate. When the incubations were carried out in the presence of 100% 2H2O, several [1-13C, 1-2H]ethanol species were detected, as well as [2-13C, 2-2H]malate and [1,3-13C2, 1,3-2H2]glycerol. The former deuterated compounds reflect the existence of NAD2H species when the incubations were carried out in 100% 2H2O, while the incorporation of 2H+ into [1,3-13C2]glycerol must be attributed to the phosphoglucose-isomerase-mediated reaction during glycolysis.

Anaerobiosis↗
PubMed1985

End products and enzyme levels of aerobic glucose fermentation in trypanosomatids.

Trypanosoma cruzi (epimastigotes), Crithidia fasciculata and Leishmania mexicana (promastigotes) were grown in a brain-heart-tryptose medium supplemented with heat-inactivated fetal calf serum. T. cruzi and C. fasciculata utilized glucose completely during the log phase of growth, whereas L. mexicana used significant amounts of the carbohydrate only at the end of the log phase and at the beginning of the stationary phase. In all cases glucose consumption resulted in excretion of succinate, and much smaller amounts of acetate. C. fasciculata and L. mexicana produced very small amounts of pyruvate. C. fasciculata produced ethanol, which was taken up again and metabolysed after glucose was exhausted. Lactate and malate were not produced. The cells were disrupted by sonic disintegration, and the activities of some key enzymes of carbohydrate and amino acid catabolism were assayed in the whole homogenates. Phosphoenolpyruvate carboxykinase was present in the three organisms; L. mexicana presented the highest specific activity. The activity of this enzyme was maximal during glucose consumption, and slightly decreased after glucose was exhausted. This suggests that the role played by the enzyme is glycolytic and not gluconeogenic; the latter is the case in most higher organisms. Hexokinase and pyruvate kinase presented their highest levels in C. fasciculata and T. cruzi during glucose consumption. L. mexicana, which was in active glycolysis during the whole experimental period, presented the highest specific activities of both enzymes. Citrate synthase, on the other hand, increased in C. fasciculata and, to a lesser extent, in T. cruzi, after glucose was exhausted; the enzyme could not be detected in L. mexicana. The NAD-linked glutamate dehydrogenase increased considerably in C. fasciculata and T. cruzi after glucose was exhausted, suggesting a catabolic role for the enzyme. This increase coincided with an increase in NH3 production by both organisms after glucose consumption. The NADP-linked glutamate dehydrogenase, on the other hand, presented a maximum about the time when glucose was exhausted, and then decreased again, which suggests a catabolic role for the enzyme. Both glutamate dehydrogenases had low activities in L. mexicana; this fits in well with the low NH3 production throughout the culture of this organism. The results are in good agreement with current ideas on the mechanism of aerobic glucose fermentation by trypanosomatids, and suggest that, under the experimental conditions used, both T. cruzi and C. fasciculata used glucose perferentially over amino acids for growth.

Animals↗
PubMed1985

Chemical modification of the active site of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi.

The NADP-linked glutamate dehydrogenase (NADP-gluDH) purified from epimastigotes of the Tulahuén strain, Tul 2 stock, of Trypanosoma cruzi, was inhibited by Cibacron Blue FG3A, and inactivated by preincubation with phenylglyoxal or Woodward's Reagent K. The inhibition by Cibracron Blue FG3A, competitive towards NADPH with an apparent Ki of 20 microM, suggests that the enzyme presents the "dinucleotide fold" characteristic of most dehydrogenases and kinases. The inactivation of the NADP-gluDH by preincubation with phenylglyoxal, with a reaction order of 1, and the partial protection afforded by alpha-oxoglutarate, suggest the presence of one arginine residue in the active site of the enzyme, which might participate in the binding of alpha-oxoglutarate through interaction with one of the carboxyl groups of the substrate. The inactivation of the NADP-gluDH by preincubation with Woodward's Reagent K suggests the presence of a carboxyl group, from an aspartic or glutamic acid residue, at the active site, which might participate in the binding of the cationic substrate NH+4. The presence of NADPH during preincubation with the reagent increased the inactivation rate, which suggests that binding of the coenzyme increases the exposure of the reactive carboxyl group.

Animals↗
PubMed1984

Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi.

The degradation of glucose by Trypanosoma cruzi leads to the excretion of succinate. Malate dehydrogenase (MDH) participates in this process by reducing to malate the oxaloacetate synthesized by the glycosomal enzyme, phosphoenolpyruvate carboxykinase. The best coupling for these two sequential reactions would be attained if both enzymes were placed in the same subcellular compartment. The intracellular distribution of the MDH activity in epimastigotes of T. cruzi was studied by two methods. Selective disruption of cellular membranes with increasing concentrations of digitonin, indicated that trypanosomal MDH is particulate. Isopycnic centrifugation in a sucrose gradient of a large granule fraction, obtained by grinding the cells with silicon carbide, showed the presence of two MDH activities: one banding together with the glycosomal marker phosphoenolpyruvate carboxykinase, the other with the mitochondrial marker citrate synthase. Isoelectrofocusing of cell-free extracts led to the separation of two enzyme forms, with pI values of about 3.5 (MDHa) and 9.4 (MDHb). These forms had similar molecular weights (approx. 60 000) and apparent Km values, but showed a small but consistent difference in their pH optima (9.23 for MDHa and 9.05 for MDHb), and in their activation by inorganic phosphate (apparent Ka values of 33 mM and 87 mM, for MDHa and MDHb, respectively). Determination of the pH optima of the enzyme forms separated by isopycnic centrifugation suggests that the glycosomal enzyme form is MDHa, and the mitochondrial one is MDHb.

Animals↗
PubMed1982

Protein glycosylation in Trypanosoma cruzi. II. Partial characterization of protein-bound oligosaccharides labeled "in vivo".

The protein-bound oligosaccharides were isolated from Trypanosoma cruzi cells incubated with [U-14C]glucose by means of as proteolytic digestion of delipidated proteinaceous pellets, followed by separation of oligosaccharides from glycopeptides through incubation with endo-N-acetylglucosaminidase H. The saccharides obtained after very short incubation periods were tentatively identified as Glc1Man9GlcNAc, Glc1Man8GlcNAc and Glc1Man7GlcNAc and the label was almost exclusively present in the glucose residues. On longer incubations, however, two more labeled oligosaccharides could be isolated, Man9GlcNAc (which appeared first) and Man9GlcNAc. The relative proportion of the three glucose-containing saccharides did not change with the length of the incubation of cells with [U-14C]glucose nor after chasing the label with the unlabeled monosaccharide. Moreover, the more internal mannose residues were labeled before in Man9GlcNAc than in Glc1Man9GlcNAc.l All this evidence indicated that Glc1Man9GlcNAc was not an intermediate in the formation of the other saccharides. On the basis of these results and of those reported in the preceding paper, the following pathway is proposed for the first stages of protein glycosylation: (formula, see text).

Animals↗
PubMed1982

Subcellular localization of phosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata.

Particulate fractions obtained from Trypanosoma cruzi and Crithidia fasciculata by different procedures were subjected to isopycnic centrifugation in sucrose gradients, in order to determine the subcellular localization of phosphoenolpyruvate carboxykinase (PEPCK) in both organisms, and of malic enzyme (ME) I in T. cruzi. The more clear-cut results were obtained with T. cruzi by breaking the cells by grinding in a mortar with silicon carbide and using a gradient from 0.4 to 2.0 M sucrose, whereas with C. fasciculata, the best procedure was disruption of the cells by digitonin treatment and potter homogenization and use of a gradient from 1.1 to 2.0 M sucrose. PEPCK banded together with the glycosomal marker hexokinase in both organisms; there was a clear separation from the mitochondrial markers, oligomycin-sensitive Mg2+-APTase and citrate synthase. PEPCK showed a latency of 24% in the enriched 'glycosoma' fraction of T. cruzi. ME I from T. cruzi, on the other hand, banded together with the mitochondrial markers. These results indicate that PEPCK and ME are present in different subcellular compartments, a fact significant for the prevention of a futile cycle between C4-dicarboxylic acids and C3-monocarboxylic acids, which might take place if both enzymes functioned in the same compartment.

Animals↗
PubMed1982

Citrate synthase from Crithidia fasciculata: inhibition by adenine nucleotides and suramin.

Citrate synthase (EC 4.1.3.7) was purified to electrophoretic homogeneity from Crithidia fasciculata ATCC 11745. 2. The purified enzyme had an optimal pH of 8.0-8.5, apparent Km values for acetyl-CoA and oxaloacetate of 5.5 and 3.5 microM, respectively, and was not activated by NH4Cl or KCl, nor inhibited by NADH or alpha-oxoglutarate. 3. Adenine nucleotides inhibited the enzyme, ATP being the most effective. The inhibition was strictly competitive towards acetyl-CoA and of the mixed type with respect to oxaloacetate. 4. The trypanocidal drug suramin inhibited both the C. fasciculata and the pig liver citrate synthases, being strictly competitive with respect to oxaloacetate, and non-competitive towards acetyl-CoA. The competitive inhibition with respect to the divalent anion oxaloacetate might be due to the strongly anionic nature of suramin, which has six sulfonic groups in its molecule.

Acetyl Coenzyme A↗
PubMed1982

Inhibition by suramin of oxidative phosphorylation in Crithidia fasciculata.

1. The ADP plus Pi-stimulated oxidation of succinate by mitochondria from the insect trypanosomatid Crithidia fasciculata was maximally inhibited (64%) by suramin at a concentration (60 microM) which did not affect the electron transport uncoupled by FCCP. Inhibition of the latter required a considerably higher concentration of the drug, 50% inhibition being attained at about 0.8 mM. 2. ATP synthesis by mitochondrial particles was inhibited by suramin, 50% inhibition being attained at about 50 microM. This inhibition was strictly competitive towards ADP, but it was not linearly competitive, since a secondary plot of apparent Km values vs concentration of the drug was strongly concave upwards. 3. The FCCP-stimulated ATPase activity of the mitochondrial particles was completely abolished either by oligomycin (20 micrograms/ml) or by 200 microM suramin. 4. The results suggest that oxidative phosphorylation may be a primary target for the trypanocide effect of suramin on organisms having, like C. fasciculata, a well-developed respiratory chain.

Adenosine Triphosphatases↗
PubMed1981

Mg2+-activated adenosine triphosphatase from Crithidia fasciculata: purification and inhibition by suramin and efrapeptin.

The mitochondrial Mg2+-activated adenosine triphosphatase (ATPase; EC 3.6.1.4) from the insect flagellate Crithidia fasciculata ATCC 11745 has been extracted from the membrane by chloroform treatment and purified to electrophoretic homogeneity by a method involving ammonium sulphate fractionation, gel filtration on Sephadex G-200 and DEAE-cellulose chromatography. The molecular weight of the native enzyme, determined by gel filtration, was about 350 000. Five subunits were detected by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, with molecular weights of 54 000, 45 000, 35 000, 20 000 and 10 000. The membrane-bound, but not the soluble (F1) ATPase was inhibited by oligomycin and leucinostatin. Both forms of the enzyme were strongly inhibited by the antibiotic efrapeptin and the trypanocidal drug suramin. The inhibition by efrapeptin was of the mixed type, with double-reciprocal plots intersecting below the abscissa, as in the case of the enzyme present in beef heart submitochondrial particles. Suramin, on the other hand, acted as a non-competitive inhibitor of the membrane-bound ATPase and as a strictly competitive inhibitor of the purified F1 ATPase.

Adenosine Triphosphatases↗
PubMed1981

Purification and regulatory properties of the NADP-linked malic enzyme for Crithidia fasciculata.

The NADP-linked malic enzyme (EC 1.1.1.40) from the insect flagellate Crithidia fasciculata has been purified to electrophoretic homogeneity by a procedure involving ammonium sulphate fractionation, gel filtration on Sephadex G-200, and column chromatography on DEAE-cellulose and hydroxylapatite. The regulatory properties of the purified enzyme have been studied, and compared with those of the two forms malic enzyme (I and II) present in Trypanosoma cruzi. The enzyme from C. fasciculata, like malic enzyme II from T. cruzi was activated by L-aspartate and succinate, which decreased the apparent Km values for both substrates, L-malate and NADP; L-aspartate in addition increased the apparent Vmax. The enzyme from C. fasciculata was inhibited by oxaloacetate, which was strictly competitive towards L-malate, with an apparent Ki (26 microM) intermediate between those reported for the two enzyme forms from T. cruzi. The C. fasciculata enzyme, like malic enzyme II from T. cruzi, was inhibited by adenine nucleotides, which were competitive towards both substrates; in addition, it was inhibited by acetyl-CoA, glyoxylate and NADH, which affected very little the activity of both enzyme froms forms T. cruzi. Thus the malic enzyme from C. fasciculata showed a regulatory pattern even more complex than that of the same enzyme from T. cruzi, despite the fact that there seems to be only one enzyme, present in the cytosol, in the insect trypanosomatid.

Animals↗
PubMed1981

Pathway of protein glycosylation in the trypanosomatid Crithidia fasciculata.

Cells of the insect parasite Crithidia fasciculata incubated with [14C]glucose were found to possess only one lipid-bound oligosaccharide with solubility in chloroform/methanol/water mixtures and net charge similar to the charges of dolichol pyrophosphate derivatives. The saccharide moiety could be released from lipid by mild acid hydrolysis. Several enzymatic and chemical treatments of the oligosaccharide indicated that the latter had the structure Man alpha leads to Man alpha leads to Man alpha leads to [Man alpha leads to Man alpha leads to Man (alpha 1 leads to 6)]Man leads to GlcNAc(beta 1 leads to 4)GlcNAc. Two labeled oligosaccharides were liberated from proteins by a sequential treatment with a protease and endo-beta-N-acetylglucosamindase H. One of the protein-bound oligosaccharides had the same structure as the lipid-linked compound, whereas in the second oligosaccharide some mannose residues had been replaced by galactose units, but both compounds migrated as did a Man7GlcNAc standard. These were the largest oligosaccharides obtained even after short labeling periods. It is suggested that glycosylation of proteins in the protozoan Crithidia fasciculata does not involved glucosylated lipid-bound oligosaccharides as intermediates.

Animals↗