Role of the NADP/thioredoxin system in the reduction of alpha-amylase and trypsin inhibitor proteins.
Thioredoxin, reduced either enzymatically with NADPH and NADP-thioredoxin reductase or chemically with dithiothreitol, reduced alpha-amylase and trypsin inhibitor proteins from several sources. Included were cystine-rich seed representatives from wheat (alpha-amylase inhibitors), soybean (Bowman-Birk trypsin inhibitor), and corn (kernel trypsin inhibitor). This system also reduced other trypsin inhibitors: the soybean Kunitz inhibitor, bovine lung aprotinin, and egg white ovoinhibitor and ovomucoid proteins. The ability of these proteins to undergo reduction by thioredoxin was determined by 1) a coupled enzyme activation assay with chloroplast NADP-malate dehydrogenase or fructose-1,6-bisphosphatase, 2) a dye reduction assay with 5',5'-dithiobis(2-nitrobenzoic acid), and 3) a direct reduction method based on the fluorescent probe, monobromobimane, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Reduction experiments with the seed proteins were carried out with thioredoxin from wheat germ (h-type) or Escherichia coli; the corresponding experiments with the animal trypsin inhibitors were carried out with thioredoxin from calf thymus or E. coli. In all cases, thioredoxin appeared to act catalytically; the reduced form of glutathione was without effect. When considered in conjunction with earlier results on purothionin (confirmed and extended in the current study), the new findings suggest that the NADP/thioredoxin system functions in the reduction of protein inhibitors of seeds and animal tissues. These results also raise the question of the occurrence of glutaredoxin in plants, as E. coli glutaredoxin was found to promote the reduction of some but not all of the proteins tested.
Kobrehel
K
K
Department of Plant Biology, University of California, Berkeley 94720.
Yee
B C
BC
Buchanan
B B
BB
D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes; D008291:Malate Dehydrogenase / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D013879:Thioredoxins / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000378:metabolism*; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005456:Fluorescent Dyes; D014361:Trypsin Inhibitors; D013879:Thioredoxins; D009249:NADP; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); D000516:alpha-Amylases