5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi

The International Journal of Biochemistry & Cell Biology 35 (2003) 1263–1271

5-Aminolevulinic acid synthesis in epimastigotes of Trypanosoma cruzi Mar´ıa Elisa Lombardo, Lidia Susana Araujo, Alcira Batlle∗ Centro de Investigaciones sobre Porfirinas y Porfirias—CIPYP (CONICET-FCEN, UBA), Ciudad Universitaria Pabellón II 2do, Piso, 1428 Buenos Aires, Argentina Received 12 February 2002; received in revised form 12 December 2002; accepted 13 December 2002

Abstract Background and aims: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. Methods: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. Results: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13 ± 0.49 EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. Conclusions: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Trypanosoma cruzi; 5-Aminolevulinic acid; Heme synthesis; 5-Aminolevulinic acid synthetase inhibition

1. Introduction Abbreviations: ALA, 5-aminolevulinic acid; ALA-S, 5-aminolevulinic acid synthetase; ALA-D, 5-aminolevulinic acid dehydratase; DOVA, 4,5-dioxovaleric acid; DOVA-T, 4,5-dioxovaleric acid transaminase; Heme-S, heme synthetase; PBG, porphobilinogen; PBGase, porphobilinogenase; PBG-D, porphobilinogen deaminase; Suc.CoA-S, succinyl coenzyme A synthetase ∗ Corresponding author. Present address: Viamonte 1881, 10 “A”, CP-1056 Buenos Aires, Argentina. Tel.: +54-11-4812-3357; fax: +54-11-4811-7447. E-mail address: [email protected] (A. Batlle).

A singular nutritional characteristic of trypanosomatid protozoa is that in vitro they need heme compounds as a growth factor, which is generally supplied as hemoglobin, hematin or hemin. The reasons for this heme nutritional requirement are not yet elucidated, and might vary among the different parasitic protozoa. However, it is widely accepted that it must be due to their inability to synthesize heme, because

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these organisms only have a partially functional heme pathway. The findings of Salzman et al. (1982); Salzman, Batlle, and De Souza (1986) would indicate that the enzymes usually confined to the cytosol, such as 5-aminolevulinic acid dehydratase (ALA-D), porphobilinogenase (PBGase), porphobilinogen deaminase (PBG-D) and probably other heme enzymes preceding heme-synthetase (Heme-S), are lacking in Trypanosoma cruzi, while the so identified as particulate enzymes, succinyl CoA synthetase (Suc. CoA-S), 5-aminolevulinic acid synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T) and Heme-S, appear to be functional as their activities could be detected. DOVA-T activity was also measured in Leishmania donovani, other member of the Trypanosomatidae family (Sagar, Salotra, Bhatnagar, & Datta, 1995). Further, Srivastava, Sharma, Kamboj, Rastogi, and Pandey (1997) have demonstrated also in L. donovani significant activities of both heme synthesizing (ALA-S and Heme-S) and degrading enzymes (heme oxygenase and biliverdin reductase). Because of the importance of heme for both the growth of the parasites and their own hemeproteins synthesis, the aim of this study has been to investigate, firstly the presence of metabolites and enzymes of heme synthesis, and then, specifically the formation of ALA, the first precursor of porphyrin pathway, in epimastigotes of T. cruzi. Moreover, the effect of a parasite acellular extract on the bacterial ALA-S activity was also investigated. 2. Materials and methods 2.1. Chemicals 4,5-Dioxovaleric acid was prepared by hydrolysis of 3,5-dibromolevulinic acid (Porphyrin Products, Logan, UT) as described by Varticovski, Kushner, and Burnham (1980). DOVA was quantified according to Jerzykowski, Winter, and Matsuszewski (1973). All other chemicals were of the highest purity commercially available. 2.2. Organisms and growth conditions T. cruzi, Tulahuén strain, Tul 2 stock (Segura et al., 1980) was supplied by the Instituto Nacional de Diag-

nóstico e Investigación de la Enfermedad de Chagas “Dr. Mario Fatala Chabén” of Buenos Aires. The cells were grown at 28 ◦ C with constant shaking in a liquid medium containing 0.3% yeast extract, 0.9% tryptose, 0.4% dextrose, 1% disodium phosphate 2-hydrate, 0.36% sodium chloride, 0.04% potassium chloride, 0.15% powered beef liver, 0.5% brain heart infusion and 0.5 mg/100 ml hemin. Rhodobacter spheroides was obtained from the collection of the Microbiology Unit, Facultad de Agronom´ıa, University of Buenos Aires. The cells were cultured in medium S of Lascelles (Lascelles, 1959), anaerobically, in the light for 2–3 days at 30 ◦ C. 2.3. Preparation of enzyme extracts from R. spheroides The harvesting of cells and preparation of cell-free extracts were carried out as described by Tuboi, Kim, and Kikuchi (1970). Nearly all the activity of ALA-S in crude extracts could be recovered in the fraction obtained at 0–40% saturation of ammonium sulfate. This fraction dialyzed overnight against 0.05 M sodium phosphate buffer pH 7.4 containing 0.01 M 2-mercaptoethanol was employed as source of ALA-S. The 45–65% ammonium sulfate fraction obtained from the same cell-free extract, was used as source of Suc.CoA-S. 2.4. Preparation of extracts from T. cruzi All manipulations steps were performed at 2–4 ◦ C. The cells were harvested in the late logarithmic phase of growth (nearly 98% epimastigotes) by centrifugation at 12,000 × g for 10 min and they were washed once with 0.05 M Tris–HCl pH 7.4 buffer. The cells from 200 ml of culture were resuspended in 8–10 ml of either 0.05 M Tris–HCl pH 7.4 buffer or 0.05 M sodium phosphate buffer pH 7.4 depending on the enzyme under study. Cells in suspension were disrupted by sonication in a Soniprep 150, MSE Ultrasonic Power for 45 s. The resulting homogenate was centrifuged at 5000 × g for 15 min and the supernatant (S) employed for measuring the intracellular levels of ALA (I-ALA), PBG (I-PBG), and porphyrins (I-porphyrins) and enzymatic activities.

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2.5. Determination of intracellular content of heme biosynthetic pathway metabolites The following determinations were carried out on cells of T. cruzi resuspended in 0.05 M Tris–HCl pH 7.4 buffer. 2.5.1. I-DOVA, I-ALA and I-PBG One milliliter sample of the S supernatant was treated with 1 ml of 10% trichloroacetic acid (TCA). The protein precipitate was separated by centrifugation and DOVA, ALA and PBG were measured in the supernatant. DOVA was quantitated by its reaction with o-phenylendiamine as described by Jerzykowski, Winter, and Matsuszewski (1973). ALA and PBG were measured as reported by Mauzerall and Granick (1956). 2.5.2. I-porphyrins One milliliter sample of the S supernatant was treated with HCl (c) to a final concentration of 5% (w/v). The protein precipitate was separated by centrifugation and porphyrins measured in the supernatant as previously described by Araujo, Lombardo, and Batlle (1994). 2.5.3. Free-heme Heme was measured after its extraction from S supernatant with acidified chloroform. The extraction system containing 3 ml of the heme solution, 1 ml of dimethyl sulphoxide (DMSO), 2 ml of 0.05 M glycine–HCl pH 2.0 buffer, 0.2 ml of 5 M NaCl and 3 ml of chloroform, was vigorously shaked, then centrifuged to separate the organic phase. Hemin was spectrophotometrically quantified by reading the absorbance at 388 nm. This procedure allows to detected hemin concentrations from 1.15 to 9.20 ␮M.

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2.7. Assay of enzymic activities 2.7.1. Suc.CoA-S The system contained 50 ␮mol Tris–HCl buffer (pH 7.4), 100 ␮mol sodium succinate, 20 ␮mol MgCl2, 0.12 ␮mol HSCoA, 5 ␮mol ATP, 10 ␮mol glutathione (reduced form), 960 ␮mol hydroxylamine (added as hydroxylamine hydrochloride–KOH mixture, pH 7.4) and S supernatant (3–5 mg protein) in a final volume of 2 ml. Incubation was carried out aerobically, in the dark, with mechanical shaking at 37 ◦ C for 30 min. The reaction was stopped by the addition of 1 ml 25% TCA: 3N HCl: 5% FeCl3 in 0.1N HCl (1:1:1) mixture. Ferric succinylhydroxamate was determined spectrophotometrically at 540 nm. 2.7.2. ALA-S The standard assay mixture consisted of 50 ␮mol Tris–HCl buffer (pH 7.4), 10 ␮mol Cl2 Mg, 2.5 ␮mol ATP, 50 ␮mol sodium succinate, 100 ␮mol glycine, 0.12 ␮mol CoA, 0.27 ␮mol pyridoxal phosphate, 3 ␮mol glutathione, 2 ␮mol EDTA, 50 ␮mol succinyl CoA synthetase suspension (1 ml suspension was sufficient to catalyze the formation of 58 ␮mol hydroxamate succinyl/h at 37 ◦ C) and enzyme preparation (3–5 or 0.15–0.20 mg protein for T. cruzi S supernatant or R. spheroides ALA-S fraction, respectively) in a total volume of 1.2 ml. Incubation was carried out aerobically, in the dark, with mechanical shaking at 37 ◦ C for 1 h. The reaction was stopped by addition of 0.3 ml 25% TCA. After centrifugation, 1 ml of supernatant was removed for ALA quantification.

The following determinations were carried out in the culture medium after harvesting the T. cruzi cells.

2.7.3. DOVA-T Conditions used were those described by Lombardo, Araujo, Juknat, and Batlle (1989). The assay system contained 100 ␮mol sodium phosphate buffer (pH 7.4), 166.67 ␮mol l-glutamate, 4.0–5.5 ␮mol DOVA and S supernatant (3–5 mg protein) in a total volume of 1 ml. Incubations were carried out aerobically, in the light, with mechanical shaking, at 37 ◦ C for 1 h. The reaction was stopped by the addition of 1 ml 10% TCA. After centrifugation, the activity was assayed by measuring the ALA formed as described above.

2.6.1. DOVA, ALA, PBG and free-heme The same methods as those above described were used.

2.7.4. ALA-D The standard incubation system contained 100 ␮mol sodium phosphate buffer (pH 7.0), 20 ␮mol 2-mercap-

2.6. Determination of extracellular content of heme pathway metabolites

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toethanol, 5 ␮mol ALA and S supernatant (3–5 mg protein) in a total volume of 1 ml. Incubations were carried out aerobically in the dark, with mechanical shaking at 37 ◦ C for 1 h. Then 0.1 ml of saturated solution of CuSO4 was added, the precipitated protein was discarded by centrifugation and the amount of PBG formed was estimated in the supernatant. 2.7.5. PBGase It was measured according to Araujo, Lombardo, Rossetti, and Batlle (1989). The standard incubation system contained 50 ␮mol sodium phosphate buffer (pH 8.0); 0.15 ␮mol PBG and S supernatant (3–5 mg protein), in a final volume of 1.5 ml. Incubations were carried out aerobically in the dark, with mechanical shaking at 37 ◦ C for 2 h. After incubation, HCl (c) was added to a final concentration of 5% (w/v) to inactivate the protein, the mixture was then illuminated with white light for 20 min to oxidize the porphyrinogens formed, the precipitate was separated by centrifugation and total porphyrins were determined in acid solution (Rimington, 1960).

Table 1 Biochemical analysis of the heme pathway in T. cruzi Accumulated metabolites (nmol) I-DOVA I-ALA I-PBG I-porphyrins Heme ALA excreted PBG excreted

ND 80.64 ± 4.45 ND ND ND 7143.96 ± 476.91 ND

Enzyme activities (EU/mg) Suc.CoA-S ALA-S DOVA-T ALA-D PBGase

434 ± 35 ND 7.13 ± 0.49 ND ND

Measurements were made as described in Section 2. Accumulated metabolites values were expressed as total nmol in 250 ml of parasite culture which yielded about 173–155 mg of protein present in 8.5 ml of S supernatant. ND: not detected.

3. Results

T. cruzi. In contrast, ALA was found in cells-free extract as well as in the culture medium of the parasite. The amount of intracellular ALA was only 1–2% of total ALA formed, indicating that the principal fate of intracellularly synthesized ALA was its excretion. Levels of extracellular ALA varied markedly with the hemin concentration present in the culture medium. For hemin concentrations of 0.33 ± 0.23 and 4.55 ± 1.46 ␮g per ml of culture, the amounts of ALA were 27.85 ± 4.03 and 6.42 ± 0.99 nmol per ml of culture, respectively). Moreover, the ALA was isolated (by selective absorption with an ion-exchange resin) and spectroscopically characterized by comparison with a standard ALA solution. On the basis of these results we would expect to find at least some activity of the ALA synthesizing enzymes. The specific activity of Succ.CoA-S was rather high and therefore non-limiting in ALA formation catalyzed by ALA-S; however, ALA-S activity was nil and the activity of DOVA-T, the other enzyme forming ALA, was low. No significant activity of ALA-D and PBGase (enzymes involved in the conversion of ALA to PBG and PBG to porphyrins, respectively) was detected either.

3.1. Metabolites and enzymes of heme biosynthetic pathway in T. cruzi

3.2. Inactivation of R. spheroides ALA-S by T. cruzi S supernatant

As shown in Table 1, DOVA, PBG, porphyrins and free heme were not detected in acellular extracts of

When ALA-S activity of R. spheroides was measured in the presence of parasite S supernatant,

2.8. Enzyme units One enzyme unit (EU) is defined as the amount of enzyme forming 1 nmol of product per hour under the standard incubation conditions. Specific activity is the number of units per mg of protein. 2.9. Statistical treatment All determinations, i.e., intracellular levels of metabolites, excretion of ALA and PBG and enzyme assays (measured with an error of 5–10%), were carried out with the same culture and the whole experiment was run three times. The absolute values did not differ by more than 10–15% from one experiment to another. Therefore, results are expressed as the mean value of three separate experiments run in duplicate ±S.D. (n = 3).

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Table 2 Gel filtration chromatography of T. cruzi S supernatant: effect of Sephadex G-25 eluted fractions on R. spheroides ALA-S activity Additions

Elution volume (ml)

Activity (%)

Protein eluate

31

100.00 ± 0.30

Non-protein eluates Fraction 1 Fraction 2 Fraction 3

56 59 62

63.84 ± 1.15 38.22 ± 0.72 68.19 ± 1.35

Parasite S supernatant was applied on a Sephadex G-25 column (1.8 cm × 68 cm), eluted with 0.05 M Tris–HCl pH 7.4 buffer and 3 ml fractions were collected. One mililiter of each fraction was added to incubation system of R. spheroides ALA-S and this enzyme activity was measured as described in Section 2. Activity in incubation standard system without additions was set as 100%. Fig. 1. Inhibition of ALA-S from R. spheroides by an acellular extract of T. cruzi. ALA-S activity was measured in dialyzed ammonium sulfate fraction (0–40% saturation) from R. spheroides in the presence of different amounts of T. cruzi S supernatant (protein concentration: 4.50 mg/ml). Experimental conditions were as described in Section 2.

inactivation occurred, suggesting that the acellular extract from T. cruzi should contain some inhibitor of ALA-S. Fig. 1 shows that inhibition of ALA-S was dependent on the volume of parasite extract added; 1 ml of S supernatant containing about 6.80 mg of protein produced 60% inhibition.

either inactivation due to its short half-life or repression of enzyme synthesis by the inhibitory factor, or both effects simultaneously. The effect of aging on the inhibitory activity of the factor was also tested. When S supernatant was stored at −20 ◦ C for longer than 24 h its the inhibitory effect began to diminish. The action of the factor as a function of incubation time is shown in Fig. 2, inhibition was observed throughout all incubation period. ALA formation of R. spheroides without adding the T. cruzi S supernatant

3.3. Properties of the ALA-S inhibitory factor present in the parasite When the T. cruzi S supernatant was previously heated at 100 ◦ C for 2 min or dialyzed during 4 h against 5 mM Tris–HCl buffer pH 7.4, no inhibition on R. spheroides ALA-S was observed. These results would suggest that a heat-unstable low molecular weight component would be responsible for the inhibitory effect. To support further this assumption the parasite S supernatant was passed through a Sephadex G-25 column and the eluted fractions were added to the incubation system of ALA-S (Table 2). Results obtained would indicate the existence of a non-protein low molecular weight inhibitor. Furthermore, in the protein excluded with the void volume, ALA-S activity was not detected (data not shown). We propose that the lack of ALA-S activity could be the result of

Fig. 2. Effect of incubation time on ALA formation. ALA-S activity of R. spheroides was measured in absence (䊊) and presence (䊉) of parasite S supernatant. Experimental conditions were as described in Section 2.

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Control (no hemin) Hemin (␮M)

Control (no DOVA) DOVA (mM)

Fig. 3. Inhibitions of ALA/S from R. spheroides by S supernatant of T. cruzi grown under different hemin concentrations. (䊊) Without added hemin, (䊉) 5 ␮g hemin per ml of culture, (䊐) 15 ␮g hemin per ml of culture. Relative growth is defined as the number of cells per ml of culture at the end of growth with respect to the number of cells per ml of culture at the beginning of growth. The relative growth obtained values were (䊊) 9.51 ± 1.08, (䊉) 22.81 ± 3.16, (䊐) 16.35 ± 1.06. Incubations conditions and ALA measurement were as indicated in legend of Fig. 1.

was found to increase linearly during the first 40 min. When ALA-S activity was measured in the presence of parasite S supernatant, linearity was observed only up to about 20 min. Finally, the inhibitory activity on ALA-S of the S supernatant of T. cruzi grown under different hemin concentrations was evaluated (Fig. 3). The higher the hemin levels the greater the inhibition. No hemin addition means the levels of hemin present in the original cells samples, which are sufficient to sustain a relative nine-fold growth rate (legend of Fig. 3). Hemin (5 ␮g/ml) was the concentration yielding the optimum growth hemin, whilst concentrations above 15 ␮g/ml clearly decreased the growth rate (data not shown). 3.4. Hemin and DOVA inhibition of R. spheroides ALA-S With the purpose of establishing the nature of T. cruzi inhibitory factor, the effect of hemin and DOVA on the enzymic formation of ALA was examined (Table 3). Hemin was prepared at a concentration of

Additions

Activity (EU)

3.95 7.92 11.87 15.83 19.80

67.65 58.65 53.17 50.60 41.76 37.08

± ± ± ± ± ±

1.58 1.19 0.08 0.40 0.71 0.15

0.3 0.5 1.0 2.0 3.0 4.0 5.0

46.94 44.20 41.00 38.94 34.11 23.09 16.01 12.45

± ± ± ± ± ± ± ±

2.00 2.61 3.01 0.16 0.56 2.17 1.08 1.50

ALA-S activity was measured as described in Section 2, except that different amounts of hemin and DOVA were added, as indicated.

30.70 ␮M; different volumes of this hemin solution, up to 1 ml, were added to the ALA-S incubation system, final concentrations attained are indicated in Table 3. It can be seen, that both hemin and DOVA exhibited a concentration dependent ALA-S inhibition.

4. Discussion Results here reported show that T. cruzi cultures were able of synthesizing and then exporting substantial amounts of ALA; instead the intracellular amounts of DOVA, PBG, porphyrins and free-heme were nil (Table 1). These findings are implying that the defect in the heme biosynthetic pathway in the parasite must lie at some enzymatic level beyond the synthesis of ALA. The finding of intracellular ALA is in agreement with Salzman et al. (1982); Salzman, Batlle, and De Souza (1986) report, however it should be noted that this is the first time that ALA is detected in the extracellular medium. The presence of ALA both intra- and extracellularly, of which most is localized outside the cell, shows a behavior similar to that previously described by Bechara’s group (Demasi, Costa, Pascual, Llesuy, & Bechara, 1997), who found that treatment of different cell cultures with ALA or succinylacetone a known potent ALA-D inhibitor, does not overload the cells with ALA, instead extracellular ALA is significantly increased. As to the

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relation between extracellular levels of ALA and hemin, it has been found that when hemin concentration was enhanced about 10 times, accumulation of ALA was diminished 4 times, these results would indirectly indicate that hemin would feed-back regulate heme synthesis by controlling ALA synthesis. As shown in Table 1, the lack of ALA-D and PBGase activities, measured in vitro, would explain the absence of both PBG and porphyrins measured in vivo, indicating that the only fate of intracellular produced ALA would be excretion. If we now analyze ALA synthesis, let us recall that there exist two pathways leading to ALA formation (Lombardo, Araujo, Juknat, & Batlle, 1988) one involving the ALA-S enzyme and the other involving the DOVA-T enzyme. According to data from Table 1, it would appear that the latter enzyme would be the responsible for the ALA accumulated extracellularly; however, because of the low specificity of DOVA-T (Foley & Beale, 1982; Noguchi & Mori, 1981; Shioi, Nagamine, & Sasa, 1984) further experiments are necessary to confirm or not this assumption. On the other hand, the apparent discrepancy between the high levels of accumulated ALA and the low activity of the ALA synthesizing enzymes (ALA-S and DOVA-T), could be attributed to the fact that enzyme activities are measured in vitro the day of harvesting the cells, while the amount of accumulated ALA is that synthesized in vivo during all the growth period of the parasite (about 5–7 days). If we quantify the amount of ALA extracellularly accumulated and the levels of the activity of the enzymes forming ALA, as a function of the growth time (between 0 and 7 days) we found that: (a) the levels of ALA gradually increase up to day 5, then stay constants up to day 7; (b) both ALA-S and DOVA-T only be measured between days 4th and 7th, while DOVA-T activity was constant along all period, ALA-S activity was undetectable. During the first 4 days activities can not be measured because there is not enough cellular mass, so it is not possible to correlate within this interval, the levels of enzyme activities with the levels of accumulated ALA (data not shown). If we would instead consider ALA-S as the enzyme synthesizing ALA in T. cruzi, its lack of detectable activity could be due to either inactivation or repression. Regarding this and without specifically looking for an inhibitor, we observed in the supernatant fraction of T.

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cruzi, the presence of a compound which had the activity of inhibiting the activity of the ALA-S which was present, as a contaminant, in the partially purified fraction of Succ.CoA-S prepared from R. spheroides (see ALA-S incubation system in Materials and Methods). We found that this acellular extract of the parasite inhibited R. spheroides ALA-S up to 60% (Fig. 1). This factor seems to be of non-protein nature, low molecular weight and heat unstable. The reaction is inhibited since the beginning of the incubation (Fig. 2), suggesting that it would not be necessary any conversion of whatever it might be to exert its action. Correlation between the concentrations of hemin used during parasite growth and changes in the inhibitory power of the S supernatant, would indicate again that heme levels modulate ALA synthesis. We found that the inhibitory factor of T. cruzi has not effect on rat liver ALA-S activity (data not shown). We considered that the specificity of such inhibitory factor should be tested on ALA-S activity from several sources. Let us consider all the possibilities to explain the inhibition of R. spheroides ALA-S activity by this T. cruzi factor. (1) Product inhibition (ALA): it has been reported that concentrations of ALA above 15 ␮M significantly inhibited ALA-S from R. spheroides, reaching to 50% at 45 ␮M ALA and nearly 100% at 90 ␮M (Viale, Wider, & Batlle, 1987). We can not attribute the inhibitory action to endogenous ALA, because, adding to the incubation system 10 ␮M ALA—a concentration of the order of endogenous ALA (8.48 ± 0.52 ␮M), inhibition was near 10–15%, much lower than that produced by the T. cruzi S supernatant containing a similar amount of ALA. (2) Heme inhibition: heme control of ALA-S operates mainly at the level of enzyme synthesis (Drew & Ades, 1989; Hamilton et al., 1991; Hamilton, Bement, Sinclair & Wetterhahn, 1988) rather than by direct inhibition of its activity. So, inhibition of ALA-S activity by its end product can not be completely discarded but if any, probabilities here are very low because endogenous accumulation of free heme would be insufficient to produce such effect. Taking into account that hemin was not detected intracellularly and that

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20 ␮M hemin is necessary to produce 40–45% inhibition of ALA-S (Table 3), it is therefore very unlikely to identify the natural inhibitor in the parasite S supernatant with hemin. (3) DOVA inhibition: as shown in Table 3, DOVA concentrations around 3 mM are needed to inhibit 50% R. spheroides ALA-S activity in vitro. DOVA might be formed endogenously by oxidation of accumulated ALA (Pereira, Curi, Kokubun, & Bechara, 1992). But the possibility that DOVA is the inhibitor should also be discarded, because: (a) DOVA was not detected intracellularly and (b) fraction 2 from the eluates of Sephadex G-25 T. cruzi S supernatant filtration (Table 2) gives negative reaction with o-phenylendiamine. In conclusion, we have shown here that the parasite is able of synthesizing ALA, but it can not be metabolized further. T. cruzi cells in culture did not accumulate the ALA produced, instead, they released ALA to the extracellular medium. By these means not any ALA toxic effects can be found either. According to these findings, we might speculate that in the parasite, the enzyme ALA-S would be controlled by at least two mechanisms. On one hand during the first stages of growth, and in the absence of heme, ALA-S would be subjected to induction. On the other hand, when heme requirement has been satisfied, the enzyme would be inhibited by some kind of factor, alike that present and partially characterized in the acellular extracts of T. cruzi. Moreover, because ALA has been found to be extracellularly accumulated, but not PBG, an additional control of heme synthesis at the level of ALA-D can not be discarded. Finally, as already discussed, lack of ALA-S activity could also be attributed to repression of enzyme synthesis. Studies to examine this possibility are now in progress.

Acknowledgements Mar´ıa Elisa Lombardo and Alcira Batlle hold the post of Scientific Researchers at the Argentine National Research Council (CONICET). Lidia Susana Araujo is a Research Fellow of the University of Buenos Aires. This work was supported by grants

from the CONICET, the UBA and the Agencia of Promoción Cient´ıfica y Tecnológica.

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