RESUMO
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+/-0.49EU/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.
Assuntos
5-Aminolevulinato Sintetase/biossíntese , Ácido Aminolevulínico/metabolismo , Trypanosoma cruzi/enzimologia , 5-Aminolevulinato Sintetase/antagonistas & inibidores , Amônia-Liases/metabolismo , Animais , Heme/metabolismo , Porfobilinogênio/metabolismo , Rhodobacter sphaeroides/enzimologia , Succinato-CoA Ligases/metabolismo , Transaminases/metabolismoRESUMO
The effect of two selenides and their selenoxides on delta-aminolevulinic acid dehydratase (delta-ALA-D) from liver of adult rats was investigated. In vivo, selenides can be oxidized to selenoxides by flavin-containing monooxygenases (FMO) and selenoxides can regenerate selenides by thiol oxidation. Phenyl methyl selenide (PhSeCH3) and 1-hexynyl methyl selenide (C4H9Ctriple bondCSeCH3) were converted to selenoxides by reaction with H2O2. PhSeCH3 and C4H9Ctriple bondCSeCH3 had no effect on delta-ALA-D up to 400 microM. Conversely, their selenoxides inhibited delta-ALA-D, and the IC(50) for enzyme inhibition was about 100 and 70 microM, respectively. Partially purified delta-ALA-D (P(55)) from swine liver was also inhibited by these selenoxides. The inhibitory action of selenoxides was antagonized by dithiotreitol (DTT). Moreover, delta-ALA-D from a plant source was inhibited by the selenoxides, suggesting a possible involvement of SH groups in a distinct site of the homologous region implicated in Zn2+ binding in mammalian delta-ALA-D. After exposure to PhSeCH3 (500 micromol/kg/day) for 45 or 30 days, the activity of delta-ALA-D from liver of mice decreased to about 50% of the control group. The in vivo inhibitory action of this compound was not antagonized by DTT. PhSeCH3 and C4H9Ctriple bondCSeCH3 had no effect on the rate of DTT oxidation, but their selenoxides oxidized DTT. The results of the present study suggest that hepatic delta-ALA-D of rodents is a potential molecular target for selenides as a consequence of their metabolism to selenoxides by FMO.
Assuntos
Sintase do Porfobilinogênio/antagonistas & inibidores , Compostos de Selênio/toxicidade , Animais , Cucumis sativus , Ditiotreitol/metabolismo , Peróxido de Hidrogênio/química , Concentração Inibidora 50 , Cinética , Fígado/enzimologia , Espectroscopia de Ressonância Magnética , Masculino , Camundongos , Oxirredução , Óxidos/síntese química , Óxidos/toxicidade , Porfobilinogênio/metabolismo , Sintase do Porfobilinogênio/química , Ratos , Ratos Wistar , Compostos de Selênio/síntese químicaRESUMO
In Saccharomyces cerevisiae, as in all eukaryotic organisms, delta-aminolevulinic acid (ALA) is a precursor of porphyrin biosynthesis, a very finely regulated pathway. ALA enters yeast cells through the gamma-aminobutyric acid (GABA) permease Uga4. The incorporation of a metabolite into the cells may be a limiting step for its intracellular metabolization. To determine the relationship between ALA transport and ALA metabolization, ALA incorporation was measured in yeast mutant strains deficient in the delta-aminolevulinic acid-synthase, uroporphyrinogen III decarboxylase, and ferrochelatase, three enzymes involved in porphyrin biosynthesis. Results presented here showed that neither intracellular ALA nor uroporphyrin or protoporphyrin regulates ALA incorporation, indicating that ALA uptake and its subsequent metabolization are not related to each other. Thus a key metabolite as it is, ALA does not have a transport system regulated according to its role.
Assuntos
Ácido Aminolevulínico/metabolismo , Transportadores de Ânions Orgânicos , Porfirinas/biossíntese , Porfirinas/metabolismo , Saccharomyces cerevisiae/metabolismo , 5-Aminolevulinato Sintetase/deficiência , 5-Aminolevulinato Sintetase/genética , 5-Aminolevulinato Sintetase/metabolismo , Ácido Aminolevulínico/farmacologia , Transporte Biológico , Ferroquelatase/genética , Ferroquelatase/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA , Genes Fúngicos/genética , Cinética , Proteínas de Membrana Transportadoras/metabolismo , Mutação/genética , Porfobilinogênio/metabolismo , Sintase do Porfobilinogênio/metabolismo , Protoporfiria Eritropoética , Protoporfirinas/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae , Uroporfirinogênio Descarboxilase/deficiência , Uroporfirinogênio Descarboxilase/genética , Uroporfirinogênio Descarboxilase/metabolismo , Uroporfirinas/metabolismoAssuntos
Inibidores Enzimáticos/farmacologia , Fígado/enzimologia , Lisina/análise , Sintase do Porfobilinogênio/química , Porfobilinogênio/metabolismo , Animais , Cinética , Ácidos Levulínicos/farmacologia , Modelos Moleculares , Sintase do Porfobilinogênio/antagonistas & inibidores , Fosfato de Piridoxal/farmacologia , Piridoxamina/análogos & derivados , Piridoxamina/farmacologia , Piruvatos/farmacologia , Ácido Pirúvico , SuínosRESUMO
The enzyme porphobilinogen deaminase (PBG deaminase, EC 4.3.1.8) catalyzes the condensation of four molecules of PBG to give the linear tetrapyrrol, hydroxymethylbilane. It has been shown that this enzyme forms stable mono-, di-, tri- and tetrapyrrole-enzyme covalent complexes. When the enzyme, partially purified in the absence or presence of phenylmethylsulfonyl fluoride (PMSF) and preincubated with PBG, was applied on DEAE-cellulose columns, three peaks with PBG deaminase activity were detected. Using Ehrlich's reagent, it was found that the active peaks corresponded to mono-, di- and tri-pyrrylmethane-enzyme complexes. Therefore, the mechanism of action of PBG deaminase from Saccharomyces cerevisiae also involves the sequential addition of four PBG units, leading to the formation of the enzyme-substrate intermediate complexes, as has already been described for the same enzyme from other sources.
Assuntos
Hidroximetilbilano Sintase/análise , Hidroximetilbilano Sintase/metabolismo , Pirróis/metabolismo , Saccharomyces cerevisiae/enzimologia , Cromatografia DEAE-Celulose/métodos , Indicadores e Reagentes , Fluoreto de Fenilmetilsulfonil , Porfobilinogênio/química , Porfobilinogênio/metabolismo , Inibidores de Proteases , Uroporfirinogênios/químicaRESUMO
Saccharomyces cerevisiae transformed with a multicopy plasmid carrying the yeast structural gene HEM2, which codes for delta-aminolevulinate dehydratase, was enriched 20-fold in the enzyme. Beginning with cell-free extracts of transformed cells, the dehydratase was purified 193-fold to near-homogeneity. This represents a 3900-fold purification relative to the enzyme activity in normal, untransformed yeast cells. The specific activity of the purified enzyme was 16.2 mumol h-1 per mg protein at pH 9.4 and 37.5 degrees C. In most respects the yeast enzyme resembles mammalian enzymes. It is a homo-octamer with an apparent Mr of 275,000, as determined by centrifugation in glycerol density gradients, and under denaturing conditions behaved as a single subunit of Mr congruent to 37,000. The enzyme requires reduced thiol compounds to maintain full activity, and maximum activity was obtained in the presence of 1.0 mM-Zn2+. It is sensitive to inhibition by the heavy metal ions Pb2+ and Cu2+. The enzyme exhibits Michaelis-Menten kinetics and has an apparent Km of 0.359 mM. Like dehydratases from animal tissues, the yeast enzyme is rather thermostable. During the purification process an enhancement in total delta-aminolevulinate dehydratase activity suggested the possibility that removal of an inhibitor of the enzyme could be occurring.
Assuntos
Sintase do Porfobilinogênio/isolamento & purificação , Saccharomyces cerevisiae/enzimologia , Concentração de Íons de Hidrogênio , Cinética , Plasmídeos , Porfobilinogênio/metabolismo , Sintase do Porfobilinogênio/genética , Sintase do Porfobilinogênio/metabolismo , Protaminas , Saccharomyces cerevisiae/genética , Compostos de Sulfidrila , Temperatura , Transformação Genética , Zinco/farmacologiaRESUMO
1. Aminolevulinate dehydratase (ALA-D) was studied in crude extract from mouse mammary carcinoma, normal mouse liver and tumour bearing mouse liver. 2. A Michaelis-Menten behaviour and Km values between 0.24 and 0.31 mM were obtained for the enzyme in either source. 3. In all three tissues there was a linear relationship between porphobilinogen formation and incubation time, up to 120 min, ALA-D was thermostable and optimum pH was at 6.8. 4. There seems to be no structural alterations in tumoural ALA-D as compared with the enzyme from liver of both normal and tumour bearing mice.
Assuntos
Neoplasias Mamárias Experimentais/enzimologia , Sintase do Porfobilinogênio/metabolismo , Animais , Estabilidade Enzimática , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Fígado/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Porfobilinogênio/metabolismoRESUMO
Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.