Lipoamide dehydrogenase from Trypanosoma cruzi: some properties and cellular localization.

Lipoamide dehydrogenase (E.C. 1.6.4.3) was found in Trypanosoma cruzi, Tulahuen strain, stocks Tul-2 and Q501, and CA-1 strain. After differential centrifugation of epimastigote homogenates, ammonium sulfate fractionation of the 105,000 g supernatant yielded a partially purified preparation which precipitated between 0.40 and 0.80 ammonium sulfate saturation. The enzyme (a) catalyzed the oxidation of dihydrolipoamide by NAD+ and the reduction of lipoamide by NADH, the forward reaction being 2.5-fold faster than the reverse reaction; (b) exhibited hyperbolic dependence on substrate concentration and (c) possessed diaphorase activity which was less than 5% of the lipoamide reductase activity. The NADH-reduced enzyme was inhibited by arsenite, cadmium and p-chloromercuribenzoate in a concentration-dependent manner. Substrate specificity allowed lipoamide dehydrogenase to be differentiated from T. cruzi trypanothione reductase and other NADPH-dependent flavoenzymes. After cell disruption, lipoamide dehydrogenase was found mostly in the cytosolic fraction and no evidence for association with the plasma membrane was obtained.