Characterization of Violaxanthin De-Epoxidase Purified in the Presence of Tween 20: Effects of Dithiothreitol and Pepstatin A (original) (raw)
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Violaxanthin de-epoxidase (VDE) is localized in the thylakoid lumen and catalyzes the de-epoxidation of violaxanthin to form antheraxanthin and zeaxanthin. VDE is predicted to be a lipocalin protein with a central barrel structure¯anked by a cysteine-rich N-terminal domain and a glutamate-rich C-terminal domain. A fulllength Arabidopsis thaliana (L.) Heynh. VDE and deletion mutants of the N-and C-terminal regions were expressed in Escherichia coli and tobacco (Nicotiana tabacum L. cv. Xanthi) plants. High expression of VDE in E. coli was achieved after adding the argU gene that encodes the E. coli arginine AGA tRNA. However, the speci®c activity of VDE expressed in E. coli was low, possibly due to incorrect folding. Removal of just 4 amino acids from the N-terminal region abolished all VDE activity whereas 71 C-terminal amino acids could be removed without aecting activity. The diculties with expression in E. coli were overcome by expressing the Arabidopsis VDE in tobacco. The transformed tobacco exhibited a 13-to 19-fold increase in VDE speci®c activity, indicating correct protein folding. These plants also demonstrated an increase in the initial rate of nonphotochemical quenching consistent with an increased initial rate of de-epoxidation. Deletion mutations of the C-terminal region suggest that this region is important for binding of VDE to the thylakoid membrane. Accordingly, in vitro lipid-micelle binding experiments identi®ed a region of 12 amino acids that is potentially part of a membrane-binding domain. The transformed tobacco plants are the ®rst reported example of plants with an increased level of VDE activity.
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When exposed to saturating light conditions photosynthetic eukaryotes activate the xanthophyll cycle where the carotenoid violaxanthin is converted into zeaxanthin by the enzyme violaxanthin de-epoxidase (VDE). VDE protein sequence includes 13 cysteine residues, 12 of which are strongly conserved in both land plants and algae. Site directed mutagenesis of Arabidopsis thaliana VDE showed that all these 12 conserved cysteines have a major role in protein function and their mutation leads to a strong reduction of activity. VDE is also shown to be active in its completely oxidized form presenting six disulfide bonds. Redox titration showed that VDE activity is sensitive to variation in redox potential, suggesting the possibility that dithiol/disulfide exchange reactions may represent a mechanism for VDE regulation.
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In the present study the interaction between the violaxanthin cycle enzyme zeaxanthin epoxidase (ZEP) and the thylakoid membrane was investigated. Isolated, active thylakoid membranes of spinach (Spinacia oleracea L.) were subjected to different salt and detergent treatments that are generally used to isolate peripheral and integral membrane proteins. These salt and detergent treatments included the use of the salts NaBr, Na 2 CO 3 and Tris and the detergents octylglucoside (OG) and dodecylmaltoside (DM). After the treatments the activity of the ZEP was determined in washed thylakoid membranes. To obtain additional information about the mode of ZEP binding to the membrane a hydrophobicity plot based on the amino acid sequence of the protein was constructed. The plot was then compared to a diagram obtained for the photosystem II antenna Lhcb1 protein whose integration into the thylakoid membrane is known. The results of the salt and detergent treatments of the thylakoid membrane suggest that the ZEP is a peripheral, rather weakly bound membrane protein. Results from the hydrophobicity plots indicate the existence of specialized protein domains which may realize the partial integration and binding of the ZEP to the thylakoid membrane.
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Vasconcellea quercifolia (Caricaceae) latex contains several cysteine endopeptidases with high proteolytic activity. Cysteine endopeptidases are the main active compounds used by the plant as a defense mechanism. A proteolytic preparation from V. quercifolia (''oak leaved papaya'') latex was purified by cation exchange chromatography. From SDS-PAGE and blotting of the selected fractions, the N-terminal amino acid sequences of polypeptides were determined by Edman's degradation. The analysis by peptide mass fingerprinting (PMF) of the enzymes allowed their characterization and confirmed the presence of seven different cysteine proteinases in the latex of V. quercifolia. Moreover, the comparison between the tryptic maps with those deposited in databases using the MASCOT tool showed that none of the isolated proteases matched with another plant protease. Notably, a propeptidase was detected in the plant latex, which is being the first report in this sense. Furthermore, the cDNA of one of the cysteine proteases that is expressed in the latex of V. quercifolia was cloned and sequenced. The consensus sequence was aligned using the ClustalX web server, which allowed detecting a high degree of identity with cysteine proteases of the Caricaceae family and establishing the evolutionary relationship between them. We also observed a high conservation degree for those amino acid residues which are essential for the catalytic activity and tridimensional structure of the plant proteases belonging to the subfamily C1A. The PMF analysis strongly suggests that the sequence obtained corresponds to the VQ-III peptidase.