INACTIVATION OF HEART DIHYDROLIPOAMIDE DEHYDROGENASE BY COPPER FENTONSYSTEMS - EFFECT OF THIOL COMPOUNDS AND METAL CHELATORS

Copper Fenton systems (Cu(II)/H2O2 and Cu(II)/Asc) inactivated the lip oamide reductase and enhanced the diaphorase activity of pig-heart lip oamide dehydrogenase (LADH). Cupric ions alone were less effective. As a result of Cu(II)/H2O2 treatment, the number of titrated thiols in L ADH decreased from 6 to 1 per subunit. NADH and ADP (not NAD(+) or ATP ) enhanced LADH inactivation by Cu(II). NADH also enhanced the effect of Cu(II)/H2O2. Dihydrolipoamide, dihydrolipoic acid, Captopril, acety lcysteine, EDTA, DETAPAC, histidine, bathocuproine, GSSG and trypanoth ione prevented LADH inactivation. 100 mu M GSH, DL-dithiothreitol, N-( 2-mercaptopropionylglicine) and penicillamine protected LADH against C u(II)/Asc and Cu(II), whereas 1.0 mm GSH and DL-dithiothreitol also pr otected LADH against Cu(II)/H2O2. Allopurinol provided partial protect ion against Cu(II)/H2O2. Ethanol, mannitol, Na benzoate and superoxide dismutase failed to prevent LADH inactivation by Cu(II)/H2O2 or Cu(II ). Catalase (native or denaturated) and bovine serum albumin protected LADH but that protection should be due to Cu binding. LADH inhibited deoxyribose oxidation and benzoate hydroxylation by Cu(II)/H2O2. It is concluded that site-specifically generated HO, radicals were responsi ble for LADH inactivation by Cu(II) Fenton systems. The latter effect is discussed in the context of ischemia-reoxygenation myocardial injur y.