PURIFICATION AND PARTIAL CHARACTERIZATION OF THIOREDOXIN REDUCTASE FROM STREPTOMYCES-AUREOFACIENS

Thioredoxin reductase (TrxR) is one of a number of flavoproteins that catalyze the transfer of electrons between pyridine nucleotides and a specific disulfide-containing substrate. Thioredoxin reductase from St reptomyces aureofaciens 3239 has been purified to homogeneity by a two -step chromatographic procedure including anion-exchange chromatograph y and affinity chromatography on 2'5'-ADP-Sepharose 4B. Molar mass det ermined by chromatography on Superose 12 HR 10/30 and sodium dodecyl s ulfate polyacrylamide gel electrophoresis revealed 69 kDa for the nati ve protein and 34.8 kDa for the enzyme subunit. The isoelectric point determined by isoelectric focusing gel electrophoresis was 4.3. TrxR e ffectively catalyzed the reduction of DTNB in the presence of S. aureo faciens thioredoxin-l. TrxR activity in the presence of S. aureofacien s thioredoxin-2 was only 1/4 of the activity with thioredoxin-1 (1). T he activity of pure TrxR decreased drastically in the presence of NADP H, while NADP(+) as well as Streptomyces aureofaciens thioredoxin-l pr otected the enzyme from inactivation. These results indicate that thio redoxin reductase activity in bacteria could be modulated by the redox status of NADP(+)/NADPH and thioredoxin pools.