DIHYDROLIPOAMIDE DEHYDROGENASE IN THE TRYPANOSOMA SUBGENUS, TRYPANOZOON

The enzyme dihydrolipoamide dehydrogenase has been discovered and char acterised in four salivarian trypanosomes of the subgenus trypanozoon: Trypanosoma brucei brucei, T.b. gambiense, T.b. rhodesiense, and Tryp anosoma evansi. The three T. brucei species, which have insect procycl ic forms biochemically distinct from their mammalian bloodstream forms , express dihydrolipoamide dehydrogenase in both cell types, but have higher levels in the procyclic forms. Determination of Michaelis const ants for the enzyme from each of the three T. brucei species did not r eveal any significant kinetic differences between the bloodstream and procyclic enzymes. On Western blots, antibodies raised against dihydro lipoamide dehydrogenase from the stercorarian trypanosome, Trypanosoma cruzi, cross-react strongly with the dihydrolipoamide dehydrogenase f rom all three T. brucei species; by this method, the relative molecula r masses of their dihydrolipoamide dehydrogenases are indistinguishabl e. Dihydrolipoamide dehydrogenase was purified from bath the bloodstre am and the procyclic forms of T.b. brucei, and the N-termini have been sequenced. These sequences are identical to the derived protein seque nce of the cloned gene (Else et al., fur. J. Biochem. 212 (1993) 423-4 29), but have a nine amino acid N-terminal truncation, giving an N-ter minus equivalent to that of T. cruzi dihydrolipoamide dehydrogenase. T he T.b. brucei dihydrolipoamide dehydrogenase gene has been expressed in Escherichia coli and the resultant protein purified; its N-terminus is processed in a similar fashion to that in the trypanosome, but wit h reduced specificity.