Berta M. Franke de Cazzulo

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.

Chromosomal localization of seven cloned antigen genes provides evidence of diploidy and further demonstration of karyotype variability in Trypanosoma cruzi

The karyotype of Trypanosoma cruzi was studied by pulsed field gel electrophoresis (PFGE) in conditions that allowed 20-25 chromosome bands to be detected. However, several of these bands were present in non-equimolar amounts, suggesting that the total chromosome number is considerably higher. The patterns obtained with the different cloned and uncloned strains were unique, suggesting that the karyotype of T. cruzi is highly variable. The chromosomal localizations of seven cloned genes were determined by Southern blotting of PFGE-separated chromosomes. Three of the clones gave rise to similar patterns and mapped on a chromosome or a family of chromosomes larger than 1.6 Mb. Two clones mapped on either single or pairs of chromosomes, which in some cases differed considerably in size between the different strains tested, suggesting that extensive chromosome rearrangements occur in T. cruzi. Another clone hybridized to several chromosomes in most strains and probably represents a family of genes. Lastly, one clone hybridized to nearly all chromosomes. Many of the clones hybridized to pairs of restriction fragments in the different strains, suggesting that they are allelic. For one of the clones it was possible to provide further evidence for the allelic nature of the fragments by establishing detailed restriction maps around them and by showing that the two fragments in a pair hybridized to chromosomes which differed slightly in size. Taken together, the results infer that the genome of T. cruzi epimastigotes is diploid.

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.

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.

Purification and some properties of an acidic protease from epimastigotes of Trypanosoma cruzi

Abstract 1. 1. An acidic protease was purified to electrophoretic homogeneity (34-fold purification, 7% yield) from epimastigotes of Trypanosoma cruzi, Tulahuen strain, Tul 2 stock. The enzyme is monomeric, with a molecular weight of about 60,000. 2. 2. The purified enzyme was able to use as substrate azocasein, casein, bovine serum albumin and hemoglobin; the highest activity was found with bovine serum albumin at pH 4.0. Soluble T. cruzi proteins were also used as substrates, with an optimum pH of about 3.0. 3. 3. The purified enzyme was rather thermostable; 50% of the enzyme activity was lost upon preincubation at 62°C for 10 min at pH 5.0. Accordingly, the “optimal” temperature for the reaction with azocasein as substrate was 60°C. 4. 4. The enzyme was strongly inhibited by the thiol reagents p-chloromercuribenzoate, phenolphthalein mercuric acetate and p-chloromercuriphenylsulfonate (I50 values of 1.0, 1.2 and 3.0 × 10−6M, respectively); thiol-containing compounds, such as 2-mercaptoethanol and reduced glutathione, activated the enzyme; the former was also able to reactivate the enzyme inhibited by the mercurials. N-α-p- Tosyl- l -lysine chloromethyl ketone and N-α-p- tosyl- l -phenylalanine chloromethylketone were also strong inhibitors (I50 of 1.2 × 10−6 and 1.7 × 10−5 M, respectively); 2-mercaptoethanol did not revert these inhibitions. 5. 5. The properties of the purified protease suggest that it may be the main factor responsible for the proteolysis of endogenous substrates that we have described in cell-free extracts of T. cruzi (Cazzulo, J. J. and Franke de Cazzulo, B. M. (1982) Experientia (Basel) 38, 1135–1137).

Pyruvate decarboxylase and alcohol dehydrogenase in the insect trypanosomatid, Crithidia fasciculata

Abstract 1. 1. Cell-free extracts of the insect trypanosomatid Crithidia fasciculata , obtained by grinding with glass powder in a mortar, contained activity of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1). Both enzymes were found in the soluble fraction (105,000 g supernatant) and were partially purified by ammonium sulphate fractionation and gel filtration on Sephadex G-200. 2. 2. Pyruvate decarboxylase, free of lactate dehydrogenase and with little endogenous alcohol dehydrogenase, was assayed spectrophotometrically with commercial yeast alcohol dehydrogenase as coupled enzyme. The activity was increased nearly 2-fold by the addition of thiamine pyrophosphate (0.1 mM) and MgCl 2 (1 mM). The apparent K m for pyruvate was 2 mM, and the optimal pH was 6.0. 3. 3. Alcohol dehydrogenase was nearly 14-fold more active in the direction of acetaldehyde reduction than in the direction of ethanol oxidation. The optimal pH was about 7.0, and the apparent K m values were 35 μM for acetaldehyde (at 0.15 mM NADH) and 14 μM for NADH (at 60 μM acetaldehyde). 4. 4. Both enzymes were strongly inhibited by thiol reagents such as p -chloromercuribenzoate and phenolphthalein mercuric acetate; pyruvate decarboxylase was the more sensitive. 5. 5. The properties of both enzymes suggest that the enzyme system for ethanol production by C. fasciculata is very similar to that present in bakers yeast.

Inhibition of the nad-linked glutamate dehydrogenase from Trypanosoma cruzi by sulfhydryl reagents

1. The NADP-linked glutamate dehydrogenase purified from epimastigotes of Trypanosoma cruzi was strongly, but not completely, inhibited by sulfhydryl reagents, in the presence of Tris-HCl or phosphate buffers. 2. The enzyme modified by preincubation with o-iodosobenzoate had a kinetic behaviour different from that shown by the enzyme modified with other inhibitors, such as N-ethylmaleimide or p-chloromercuribenzoate. 3. The inhibition by o-iodosobenzoate was additive with the inhibition by the other reagents tested. 4. It is suggested that two or more different sulfhydryl groups, placed probably near the active site, are involved in these effects.

Inhibition by suramin of the glutamate dehydrogenases from Trypanosoma cruzi and bovine liver

Abstract 1. 1. The NAD-linked (EC 1.4.1.2) and the NADP-linked (EC 1.4.1.4) glutamate dehydrogenases from Trypanosoma cruzi , and the coenzyme-unspecific (EC 1.4.1.3) glutamate dehydrogenase from bovine liver, were inhibited by the trypanocidal drug suramin. 2. 2. The mammalian enzyme was the most sensitive (I 50 , concentration of inhibitor for 50% inhibition, of 3 μM), followed by the NAD-linked enzyme from T. cruzi (I 50 of 18 μM). The NADP-linked enzyme from T. cruzi was maximally inhibited by about 28% by 50 μM suramin. 3. 3. Suramin was a non-competitive inhibitor with respect to the substrates α-oxoglutarate, NH 4 Cl and l -glutamate in all cases, and with respect to NAD(H) and NADP(H) in the cases of the mammalian enzyme and the NAD-linked glutamate dehydrogenase from T. cruzi . 4. 4. On the other hand, the inhibitor was strictly competitive towards NADPH and NADP in the case of the NADP-linked enzyme from T. cruzi .

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.