TY - JOUR
T1 - Characterization of violaxanthin de-epoxidase purified in the presence of Tween 20
T2 - Effects of dithiothreitol and pepstatin A
AU - Kuwabara, Tomohiko
AU - Hasegawa, Mika
AU - Kawano, Mitsuko
AU - Takaichi, Shinichi
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1999
Y1 - 1999
N2 - 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 41kDa 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).
AB - 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 41kDa 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).
KW - Dithiothreitol
KW - Pepstatin A
KW - Spinach
KW - Tween 20
KW - Violaxanthin de-epoxidase
KW - Xanthophyll cycle
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U2 - 10.1093/oxfordjournals.pcp.a029496
DO - 10.1093/oxfordjournals.pcp.a029496
M3 - Article
C2 - 10635115
AN - SCOPUS:0033227476
VL - 40
SP - 1119
EP - 1126
JO - Plant and Cell Physiology
JF - Plant and Cell Physiology
SN - 0032-0781
IS - 11
ER -