In vitro assays with recombinant VDL and VDE proteins under standard conditions (31, 32) were carried out in reaction buffer at pH 5.2 containing 40 mM MES [2-(N-morpholino)ethanesulfonic acid], 10 mM KCl, and 5 mM MgCl2 and a final volume of 1 ml. Ten microliters of a 40 μM ethanolic stock solution of the respective carotenoid substrate and 10 μl of a 1.16 mM methanolic MGDG (monogalactosyl diacylglycerol; Lipid Products) stock were thoroughly mixed in a 1.5-ml reaction tube, followed by rapid addition of 920 μl of reaction buffer and vortexing for 10 s. For VDE assays and where explicitly stated for VDL assays, 20 μl of 1.5 M aqueous sodium ascorbate was added. The reaction was started by the addition of 60 μl of enzyme in renaturation buffer and vortexing for 10 s. Reaction tubes were incubated in a water bath at 20°C. Reactions were stopped by the addition of 300 μl of 1 M NaOH and incubation on ice. For determination of PtVDL1 reaction kinetics, assay volume was scaled up to 8 ml, and sample volume per time point was 1 ml. Pigment/lipid aggregates were harvested by centrifugation (18,000g, 2 min), the supernatant was removed, and the pellets were incubated at −20°C at least 15 min but not more than 24 hours until pigment extraction for high-performance liquid chromatography (HPLC) analysis.

For determination of the pH dependence of PtVDE and PtVDL1, the pH of the reaction buffer was adjusted with NaOH or HCl. Three independent experiments each covering the range of examined pH values were performed. PtVDL1 activity was estimated from the amount of violaxanthin converted to neoxanthin after 10 min, while PtVDE activity was determined from the amount of diadinoxanthin converted to diatoxanthin after 5 min. To account for minor activity differences between the three experiments, each dataset was fitted using an extreme value distribution function [y = a*e^(−e^[−((xb)/c)] − ((xb)/c) + 1); Eqn. 8033 in TableCurve 2D version 4; AISN Software], the maximum of the curve fit was set as 100%, and the measured activity data were normalized to this value. In Fig. 4 (C and F), the combined data were plotted against pH and a curve fit with Eqn. 8033 including all data points added.

For determination of DTT sensitivity, 5 M DTT in reaction buffer was added to the assays to attain final concentrations between 0.05 and 500 mM DTT. Enzymes were incubated with DTT for 10 min before reactions were started. Three independent experiments each covering the range of examined DTT concentrations including controls without DTT were performed. Enzyme activities were normalized to the activities of controls without DTT (=100%), and the combined activity data were plotted against DTT concentrations (Fig. 4, D and G). Dose-response curves of enzyme inhibition by DTT were calculated from the data using a three-parameter logistic function [y = a/(1 + (x/b)^c); Eqn. 8076 in TableCurve 2D].

For determination of the ascorbate dependence of PtVDL1 reaction kinetics, the assay volume was scaled to 5 ml. The assay was started by the addition of the enzyme already solubilized in buffer to the MGDG/violaxanthin solution. After vortexing for 5 s, the 5-ml assay was split immediately in two 2.5-ml aliquots that were pipetted to 50 μl of 1.5 M aqueous sodium ascorbate or 50 μl of demineralized water. At each time point, samples of 350-μl volume were withdrawn in parallel from the two aliquots, transferred to reaction vessels on ice and containing 100 μl of 1 N NaOH, and pigments were analyzed by HPLC as described above. Pigment analysis was performed by HPLC. The kinetics of violaxanthin decrease were fitted using an equilibrium concentration function [y = (ab)*e^(−c*t) + b; Eqn. 8143 in TableCurve 2D]. For determination of the ascorbate dependence of PtVDE, an assay volume of 2 ml containing MGDG/violaxanthin and the enzyme solubilized in buffer was split in two 1-ml aliquots and either 20 μl of 1.5 M aqueous sodium ascorbate or 20 μl of demineralized water added. After 10 min, samples of 350-μl volume were withdrawn in parallel from the two aliquots and further processed as described for determination of the ascorbate dependence of PtVDL1.

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