Characterization of violaxanthin de-epoxidase purified in the presence of Tween 20: effects of dithiothreitol and pepstatin A.
Violaxanthin de-epoxidase (VDE) was purified from thylakoid membranes of spinach by conventional column chromatography in the presence of Tween 20. The neutral detergent was necessary to prevent non-specific interaction of VDE with column resins. In anion-exchange chromatography on Mono Q, VDE appeared in two peaks. Both peaks exhibited a polypeptide of 41 kDa when fully reduced with 5 mM dithiothreitol. Re-chromatography of either peak gave rise to both peaks, suggesting that the two forms of VDE are interconvertible. VDE characteristically changed its electrophoretic mobility depending on the concentration of dithiothreitol with which the protein was treated. When non-reduced, it showed two polypeptides of 43 and 42 kDa. These polypeptides moved down to the position of 40 kDa, and then up to the position of 41 kDa, along with the increase in the dithiothreitol concentration from 0 to 2 mM. These findings suggest that VDE has more than one disulfide bond and takes multiple forms depending on the extent of the reduction. Studies with various types of protein-modifying reagent revealed that VDE is sensitive to pepstatin A, a specific inhibitor of aspartic protease. This finding suggests that the reaction center of VDE contains a reactive aspartic acid residue(s).
Kuwabara
T
T
Institute of Biological Science, University of Tsukuba, Japan.
Hasegawa
M
M
Kawano
M
M
Takaichi
S
S
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010436:Pepstatins / Q000737:chemistry*; D011136:Polysorbates
D010436:Pepstatins; D011136:Polysorbates; C014195:Streptomyces pepsin inhibitor; D010088:Oxidoreductases; C023290:violaxanthin de-epoxidase; D004229:Dithiothreitol; C031375:pepstatin