Heme synthetase in Trypanosoma cruzi.

Heme-synthetase (Heme-S) has been studied in the epimastigote form of T. cruzi (Tulahuen and Y strains). The enzyme is confined to the "mitochondrial" fraction (sedimented at 30,000 g). Activity was dependent on protein and time of cell storage. Enzymic proto- and meso-heme formation was inhibited up to 40 and 72% respectively by Triton X-100. The optimum pH was 7.2 for protoheme and 7.5 for mesoheme formation. Heme-S reached its maximum when the concentration values were 37; 35 and 32 microM for proto-, meso-, and deuteroporphyrin, respectively. The activity is several times higher when mesoporphyrin is used as substrate. At a final conc of 100 microM Fe2+ and Zn2+ ions enhanced activity 200-400%. Cu2+ and Co2+ had no effect, while Mn2+ and Mg2+ were highly inhibitory. A combination of Fe2+ and Zn2+ at varying concentrations still showed great activation. However, at a fixed level of Fe2+, Cu2+ was changed into a strong inhibitor. We propose that, if it can be demonstrated that T. cruzi cannot multiply when protoheme is replaced by mesoheme, administration of mesoporphyrin would then greatly affect replication of T. cruzi. Furthermore, the addition of certain metals, such as Cu2+ to T. cruzi cultures might specifically inhibit the parasite enzyme opening the possibility of selectively destroying the hemoflagellate without affecting the host.

Heme synthesis in Trypanosoma cruzi: influence of the strain and culture medium.

The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in two strains of Trypanosoma cruzi (Y and CL) grown in two culture media (LIT and Warren): succinyl coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. ALA and PGB were detected in both strains of T. cruzi. However, ALA was not detected in epimastigotes of the Y strain grown in the LIT medium. The content of ALA and PBG varied according to the strain and the growth medium. No free porphyrins and heme were detected in both strains of T. cruzi. The activity of Suc.CoA-S and DOVA-T was markedly influenced by the strains of the parasite and the growth medium. No significant DOVA-T activity was detected in epimastigotes of the CL strain grown in the Warren's medium. No significant activity of ALA-D, PBGase and deaminase was detected in T. cruzi. Activity of Heme-S was detected in both strains of T. cruzi when mesoporphyrin, protoporphyrin or deuteroporphyrin was used as substrate. The enzyme activity was influenced by the strain of the parasite, the growth medium and the substrate used.

Heme synthesis in Crithidia deanei: influence of the endosymbiote.

The activity of the following enzymes involved in the biosynthesis of porphyrins was determined in endosymbiote-free and endosymbiote-containing Crithidia deanei grown in a chemically defined medium: succinyl Coenzyme A synthetase (Suc.CoA-S), 5-aminolevulinate synthetase (ALA-S), 4,5-dioxovaleric acid transaminase (DOVA-T), 5-aminolevulinate dehydratase (ALA-D), porphobilinogenase (PBGase), deaminase and heme synthetase (Heme-S). The amount of 5-aminolevulinic acid (ALA) and porphobilinogen, porphyrins and heme was also determined. ALA and PBG were detected in C. deanei. The levels of free porphyrins was low. Heme concentration was nil. The activity of ALA-D, deaminase and PBGase was not detected in C. deanei. The activity of Suc.CoA-S and ALA-S were twice higher in symbiote-containing than in aposymbiotic C. deanei. Aposymbiotic cells had a higher activity of DOVA-T than symbiote-containing cells. The level of Heme-S, measured using protoporphyrin as substrate, was twice as high in symbiote-containing than in symbiote-free cells.

Porphyrin biosynthesis in parasitic hemoflagellates: functional and defective enzymes in Trypanosoma cruzi.

1. Heme compounds are necessary as a growth factor for Trypanosoma cruzi in culture, this porphyrin requirement being due to the inability of the parasite to synthesize heme. To obtain supporting evidence for this hypothesis, an extensive study of porphyrin biosynthesis in the epimastogote form of T. cruzi (Tulahuén strain) was carried out. 2. Low levels of endogenous delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) were found in extracts of T. cruzi. Free porphyrins and heme contents were practically nil. 3. The activity of succinyl CoA synthetase (Suc. CoA-S) was rather high and therefore non-limiting. 4. Both delta-aminolevulinic acid synthetase (ALA-S) and 4.5, dioxovaleric transaminase (DOVA-T), the two enzymes forming ALA, were readily detected and their activities, although low, were of the same order. 5. delta-Aminolevulinic acid dehydratase (ALA-D) activity was almost negligible and both porphobilinogenase (PBGase) and deaminase were absent or inactive. 6. Heme-Synthetase (Heme-S) was totally functional. 7. It is concluded that T. cruzi has lost part of its heme biosynthetic pathway, possibly due to mutations of several genes involved in the synthesis of the soluble enzymes ALA-D, PBGase, deaminase and probably others preceding Heme-S; while the particulate enzymes Suc CoA-S, ALA-S, DOVA-T and Heme-S are functional. As a consequence, the host should supply the parasite with the porphyrin substrate to form its essential heme compounds.