Purification and partial characterization of two thioredoxins from Streptomyces aureofaciens.
Thioredoxins are low molecular weight proteins, which participate in a wide spectrum of biochemical reactions. Two thioredoxins from Streptomyces aureofaciens 3239 have been purified to homogeneity by a sequence of chromatography steps including chromatography on Sephacryl S-300, Phenyl Sepharose CL 4B and MonoQ HR 5/5. Thioredoxin activity clearly separates into two protein fractions on MonoQ HR 5/5 chromatography. Molecular weights determined by chromatography on Superose 12 HR 10/30 and sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed M(r) approximately 10,500 for thioredoxin 1 (TR1) and M(r) approximately 11,000 for thioredoxin 2 (TR2). The isoelectric points of the two thioredoxins are different pI = 4.7 for TR1 and 5.6 for TR2, respectively. Both were effectively reduced with NADPH in reaction catalyzed by Streptomyces aureofaciens thioredoxin reductase. The specific activity of viewly for discovered TR2 is about 1/4 of the specific activity of TR1. Both thioredoxins activate spinach NADPH-malate dehydrogenase. Activation of this enzyme by TR2 is only half effective than by TR1. The stability of TR1 is high and similar to thioredoxins from other organisms unlike the activity of TR2 which is decreased during purification. The proteins diversed in their contents in exponentially growing mycelium.
Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Bratislava, Slovak Republic.
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T
Horecká
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Perecko
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Kutejová
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K
Muchová
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M
Kollárová
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007526:Isoelectric Point; D008291:Malate Dehydrogenase / Q000378:metabolism; D008970:Molecular Weight; D018724:Spinacia oleracea / Q000201:enzymology; D013304:Streptomyces aureofaciens / Q000737:chemistry*; D013879:Thioredoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism
D001426:Bacterial Proteins; D013879:Thioredoxins; D008291:Malate Dehydrogenase; C031917:malate dehydrogenase (oxaloacetate-decarboxylating) (NADP+)