Jg. Correa et Aom. Stoppani, INACTIVATION OF HEART DIHYDROLIPOAMIDE DE SHYDROGENASE BY CU(II) AND HYDROGEN-PEROXIDE OR ASCORBATE, Medicina, 54(4), 1994, pp. 319-330
The inactivation of pig-heart dihydrolipoamide (LipDH) by oxy-radicals
generated by Cu(II), supplemented or not with hydrogen peroxide (Fent
on system-Cu(II): SF-Cu(II) or ascorbate (Cu(II)-Asc), was studied. Th
e reagents concentrations used were 2,5-10 mu M Cu(II): 3.0 mM H2O2, a
nd 0,5 mM ascorbate. After 5 minutes incubation at 30 degrees, LipDH a
ctivity was measured as described by Gutierrez Correa and Stoppani (Re
ference 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 act
ivity increased several-fold (Table 1), thus showing a site-specific d
amage of LipDH thiols. NAD(+), dihydrolipoamide, GSSG, CAPTO-PRIL, met
al chelators (L-histidine, bathocuproine, EDTA, DETAPAC), trypanothion
e and allopurinol) protected LipDH from inactivation by SF-Cu(II) (Tab
les 2, 4-6). The same compounds, GSH, dithiothreitol, N-acetylcysteine
, mercaptopropionylglycine and DL-penicillamine protected the enzyme f
rom 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 (Ta
ble 8) enhanced LipDH inactivation by the SFCu(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, et
hanol) 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 radical
s and its modification by therapeutic agents are discussed in the cont
ext of the physiopathology of heart injury after post-ischemic reoxyge
nation.