Inactivation of Trypanosoma cruzi dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase systems: role of hypochloride and nitrite related radicals.

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.

Inactivation of Trypanosoma cruzi dihydrolipoamide dehydrogenase by leukocyte myeloperoxidase systems: role of hypochloride and nitrite related radicals

Dihydrolipoamide dehydrogenase (LADH) from Trypanosoma cruzi, the causative agent of Chagas' disease, was inactivated by treatment with myeloperoxidase (MPO)-dependent systems. LADH lipoamide reductase and diaphorase activities decreased as a function of incubation time and composition of the MPO/H2O2/halide system, a transient increase preceding the loss of diaphorase activity. Iodide, bromide, thiocyanide and chloride were effective components of MPO/H2O2 or MPO/NADH systems. Catalase prevented LADH inactivation by the MPO/NADH/halide systems in agreement with H2O2 production by NADH-supplemented LADH. Thiol compounds (L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine) and Captopril prevented LADH inactivation by the MPO/H2O2/NaCl system and by NaOCl, thus supporting HOCl as agent of the MPO/H2O2/NaCl system. MPO/H2O2/NaNO2 and MPO/NADH/NaNO2 inactivated LADH, the reaction being prevented by MPO inhibitors and thiol compounds. T. cruzi LADH was affected by MPO-dependent systems like myocardial LADH, allowance being made for the variation of the diaphorase activity and the greater sensitivity of the T. cruzi enzyme to MPO/H2O2/halide systems.

Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds.

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H2O2/halide, MPO/NADH/halide and MPO/H2O2/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H2O2 system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H2O2/NaCl system. NaOCl and the MPO/H2O2/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H2O2 production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horse-radish peroxidase supplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H2O2/NaNO2 system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated "reactive species" oxidize essential thiol groups at LADH catalytic site.

[Inactivation of myocardial dihydrolipoamide dehydrogenase by Cu(II) and hydrogen peroxide]. / Inactivación de la dihidrolipoamida deshidrogenasa de miocardio por Cu(II) y peróxido de hidrógeno.

The inactivation of pig-heart dihydrolipoamide (LipDH) by oxy-radicals generated by Cu(II), supplemented or not with hydrogen peroxide (Fenton system-Cu(II): SF-Cu(II)) or ascorbate (Cu(II)--Asc), was studied. The reagents concentrations used were 2.5-10 microM Cu(II): 3.0 mM H2O2, and 0.5 mM ascorbate. After 5 minutes incubation at 30 degrees, LipDH activity was measured as described by Gutiérrez Correa and Stoppani (Reference 13). As a result of peroxide effect, LipDH lipoamide reductase activity decreased in most cases by 83-98% (with the SF-Cu(II) and Cu(II)-Asc system) or 46-53% with Cu(II) only. The enzyme diaphorase activity increased several-fold (Table 1), thus showing a site-specific damage of LipDH thiols. NAD+, dihydrolipoamide, GSSG, CAPTO-PRIL, metal chelators (L-histidine, bathocuproine, EDTA, DETAPAC), trypanothione and allopurinol) protected LipDH from inactivation by SF-Cu(II) (Tables 2, 4-6). The same compounds, GSH, dithiothreitol, N-acetylcysteine, mercaptopropionylglycine and DL-penicillamine protected the enzyme from inactivation by Cu(II) (Tables 2, 4-6). L-cysteine only protected from Cu(II), to a limited degree (Table 4). Compounds protecting LipDH did not reactivate the inactivated enzyme (Table 7). NADH (Table 2), OH-DOPAMINE, DOPA, dihydroxy-phenylacetic acid (DOPAC) and catechol (Table 8) enhanced LipDH inactivation by the SF-Cu(II) but not by Cu(II), except OH-dopamine. ATP and ADP enhanced LipDH inactivation by Cu(II), but not by SF-Cu(II) (Table 3). HO scavengers (benzoate, mannitol, ethanol) and superoxide dismutase did not prevent LipDH inactivation by Cu(II) and H2O2. Catalase protected but its action was not related to its catalytic activity (Table 9). LipDH inactivation by oxygen radicals and its modification by therapeutic agents are discussed in the context of the physiopathology of heart injury after post-ischemic reoxygenation.