[Inactivation of myocardial dihydrolipoamide dehydrogenase by Cu(II) and hydrogen peroxide].
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.
Gutiérrez Correa
J
J
Centro de Investigaciones Bioenergéticas, Facultad de Medicina, Universidad de Buenos Aires, Argentina.
Stoppani
A O
AO
D000818:Animals; D003300:Copper / Q000378:metabolism / Q000494:pharmacology*; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D015428:Myocardial Reperfusion Injury / Q000378:metabolism; D013552:Swine
D003300:Copper; D006861:Hydrogen Peroxide; D008058:Dihydrolipoamide Dehydrogenase
Inactivación de la dihidrolipoamida deshidrogenasa de miocardio por Cu(II) y peróxido de hidrógeno.