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Title: Purification and characterization of violaxanthin de-epoxidase, an enzyme of the xanthophyll cycle 
Author: Rockholm, David C
Date: 1995
Abstract: Violaxanthin de-epoxidase (VDE) catalyzes the conversion of violaxanthin to zeaxanthin in chloroplast thylakoids in the presence of ascorbate and an acidic lumen. In higher plants zeaxanthin plays a role in photoprotection by mediating non-radiative (heat) dissipation of light energy in the light-harvesting complexes of photosystem II (PSII) whenever the light intensity exceeds a plant's capacity for carbon-fixation. Because of VDE's central role in this process, its purification and characterization are of interest. VDE had been partially purified from Lactuca sativa var. Romaine by differentially extracting sonicated thylakoids at both neutral and acid pH's, followed by size exclusion chromatography. I have now purified VDE by anion-exchange chromatography (Pharmacia Mono Q column) and a unique lipid-affinty precipitation step to one major polypeptide detectable by two-dimensional 50S-PAGE. VDE at pH 5.20 associates with monogalactosyl diacylglycerol (MGOG), the principal thylakoid lipid, forming a complex that can be precipitated by ultracentrifugation. In contrast to MGDG, very little VDE precipitated in the presence of eight other lipids, indicating a specific association of VDE for MGDG. The uniqueness of VDE's affinity for MGDG is further exemplified through a negative result: established" protein purification procedures using various surfactants to form reverse micelles failed to isolate VDE. The purified enzyme has an apparant molecular mass of 43 kD and a pI of 5.4. The KM values of VDE for its substrates were 0.352 µM for violaxanthin and 8.54 mM for ascorbate. Neutral red, 9-aminoacridine, and dibucaine (substances used to determine pH intracellularly and/or selectively uncouple the lumen proton gradient) directly inhibited VDE. VDE's amino acid composition and a number of partial amino add sequences from VDE were determined (its N-terrninus and four tryptic fragments). Various diverse characteristics of the purified enzyme are also reported, including how TO and BSA affect VDE activity and stability as well as the absence of mobility shifts on polyacrylamide gels. Polyclonal antibodies were generated to purified VDE (in conjunction with Katrin Hinderhofer)which inhibited the de-epoxidase reaction and reacted principally to the 43 kD polypeptide on a Western blot.
Description: Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. Includes bibliographical references (leaves 128-145). Microfiche. xvii, 145 leaves, bound ill. 29 cm
URI: http://hdl.handle.net/10125/9479
Rights: All UHM dissertations and theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission from the copyright owner.
Keywords: Xanthophylls, Carotenoids

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