Characterization of cyanobacterial ferredoxin-NADP+ oxidoreductase molecular heterogeneity using chromatofocusing.
Chromatofocusing has been used as an analytical tool to check preparations of the enzyme ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) purified in either the presence or absence of the serine protease inhibitor phenylmethylsulfonyl fluoride from the cyanobacterium Anabaena sp. strain 7119. Only one isoelectric species was found when the crude extract was processed in the presence of the protease inhibitor. Nevertheless, when the inhibitor was omitted, four ionic forms of the enzyme--showing apparent pI's in the range 4.3-4.6--were separated after chromatofocusing of the purified preparation. These forms were found to differ in their specific activities, exhibiting, on the other hand, lower values than the single one obtained in the presence of the protease inhibitor. Analysis by acrylamide gel electrophoresis revealed virtually a single main protein band except for the ionic form of pI 4.39, which was clearly resolved into two active components. Except for the more basic form, which seems to be an homodimer of Mr 80,000, all the protein components were found to be monomeric species in the range Mr 33,000-38,000. These results indicate that the molecular heterogeneity of the ferredoxin-NADP+ oxidoreductase purified from the cyanobacterium Anabaena sp. strain 7119 may result from the activity of a protease present in the whole cell homogenates. On the other hand, these data also point out that chromatofocusing should be considered as an effective technique in the isolation and characterization of the different molecular forms of this enzyme.
D000596:Amino Acids / Q000032:analysis; D002845:Chromatography / Q000379:methods; D000458:Cyanobacteria / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D005287:Ferredoxin-NADP Reductase / Q000032:analysis*; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000032:analysis*
D000596:Amino Acids; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases