Purification and characterization of lipoamide dehydrogenase from Trypanosoma cruzi.
From Trypanosoma cruzi, the causative agent of Chagas' disease, a lipoamide dehydrogenase was isolated. The enzyme, an FAD-cystine oxidoreductase, shares many physical and chemical properties with T. cruzi trypanothione reductase, the key enzyme of the parasite's thiol metabolism. 1. From 60 g epimastigotic T. cruzi cells, 2.7 mg lipoamide dehydrogenase was extracted. The flavoenzyme was purified 3000-fold to homogeneity with an overall yield of 26%. 2. The enzyme is a dimer with a subunit Mr of 55,000. With 1 mM lipoamide (Km approximately 5 mM) and 100 microM NADH (Km = 23 microM), the specific activity at pH 7.0 is 297 U/mg. 3. With excess NADH, the enzyme is reduced to the EH2.NADH complex and, by addition of lipoamide, it is reoxidized, indicating that it can cycle between the oxidized state E and the two-electron-reduced state, EH2. 4. As shown by N-terminal sequencing of the enzyme, 21 out of 30 positions are identical with those of pig heart and human liver lipoamide dehydrogenase. The sequenced section comprises the GGGPGG stretch, which represents the binding site for the pyrophosphate moiety of FAD. 5. After reduction of Eox to the two-electron-reduced state, the enzyme is specifically inhibited by the nitrosourea drug 1,3-bis(2-chloroethyl)-1-nitrosourea (Carmustine), presumably by carbamoylation at one of the nascent active-site thiols. 6. Polyclonal rabbit antibodies raised against T. cruzi lipoamide dehydrogenase and trypanothione reductase are specific for the respective enzyme, as shown by immunoblots of the pure proteins and of cell extracts.
Lohrer
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Institut für Biochemie II, Universität Heidelberg, Federal Republic of Germany.
Krauth-Siegel
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RL
D000595:Amino Acid Sequence; D000818:Animals; D002330:Carmustine / Q000494:pharmacology; D002846:Chromatography, Affinity; D008058:Dihydrolipoamide Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry; D009994:Osmolar Concentration; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D009247:NADH, NADPH Oxidoreductases; C049608:trypanothione reductase; D008058:Dihydrolipoamide Dehydrogenase; D002330:Carmustine