The constitutive xanthine dehydrogenase and the inducible 2-furoyl-coenzyme A (CoA) dehydrogenase could be labeled with ['85W]tungstate. This labeling was used as a reporter to purify both labile proteins. The radioactivity cochromatographed predominantly with the residual enzymatic activity of both enzymes during the first purification steps. Both radioactive proteins were separated and purified to homogeneity. Antibodies raised against the larger protein also exhibited cross-reactivity toward the second smaller protein and removed xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase activity up to 80 and 60% from the supernatant of cell extracts, respectively. With use of cell extract, Western immunoblots showed only two bands which correlated exactly with the activity stains for both enzymes after native polyacrylamide gel electrophoresis. Molybdate was absolutely required for incorporation of '85W, formation of cross-reacting material, and enzymatic activity. The latter parameters showed a perfect correlation. This evidence proves that the radioactive proteins were actually xanthine dehydrogenase and 2-furoyl-CoA dehydrogenase. The apparent molecular weight of the native xanthine dehydrogenase was about 300,000, and that of 2-furoyl-CoA dehydrogenase was 150,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of both enzymes revealed two protein bands corresponding to molecular weights of 55,000 and 25,000. The xanthine dehydrogenase contained at least 1.6 mol of molybdenum, 0.9 ml of cytochrome b, 5.8 mol of iron, and 2.4 mol of labile sulfur per mol of enzyme. The composition of the 2-furoyl-CoA dehydrogenase seemed to be similar, although the stoichiometry was not determined. The oxidation of furfuryl alcohol to furfural and further to 2-furoic acid by Pseudomonas putida Ful was catalyzed by two different dehydrogenases.Pseudomonas putida Ful was isolated from an enrichment culture with 2-furoic acid as the sole source of carbon and energy (24). The formation of 2-furoyl-coenzyme A (CoA) is the first reaction step in 2-furoic acid degradation. The second enzymatic step is catalyzed by a dehydrogenase that introduces a hydroxyl group to form 5-hydroxy-2-furoylCoA (23,24,49,50). Other substrates for P. putida Ful were furfuryl alcohol and furfural, which were degraded via 2-furoic acid and 2-furoyl-CoA (Fig.

Trypanothione, the essential metabolite in the oxidant defense system of trypanosomatids, is synthesized by two distinct proteins, glutathionylspermidine synthetase and trypanothione synthetase. Glutathionylspermidine synthetase was purified to homogeneity from the trypanosomatid Crithidia fasciculata by aqueous two-phase systems and chromatography. The enzyme showed a specific activity of 38 mol of glutathionylspermidine formed per min per mg of protein. Its molecular mass was 78 kDa in SDS-polyacrylamide gel electrophoresis, and it appeared predominantly monomeric in native polyacrylamide gel electrophoresis and gel filtration. The isoelectric point was at pH 4.6, and the pH optimum was near 7.6. Partial amino acid sequencing revealed homology with, but low similarity to, the glutathionylspermidine synthetase/amidase of Escherichia coli, and amidase activity was not detected in glutathionylspermidine synthetase of C. fasciculata.

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