Reaction System for Violaxanthin De-Epoxidase with PSII Membranes

PSII membranes were used as a substrate for violaxanthin de-epoxidase(VDE) that had been solubilized from spinach thylakoids by sonication.Inclusion of Tween 20 in the assay mixture was essential, althoughthe detergent apparently inhibited the activity in the conventionalassay with purified violaxanthin and lipid as substrate. Themaximum enhancing effect of the detergent was observed nearits critical micellar concentration. It is likely that the monomerof the detergent helped VDE react with the substrate in themembranes. Dependence of the activity on the substrate concentrationsuggested that VDE functions at least at two sites in the membranes,probably on both their lumenal and stromal surfaces. The abilityof the enzyme to function on the stromal surface in in vitroassays was demonstrated by using intact thylakoids as the substrate.Under such conditions where the endogenous VDE was functioningin the lumen, the exogenously added VDE converted an-theraxanthinto zeaxanthin in the absence of Tween 20. This result suggeststhat, in the reaction with PSII membranes, the detergent wasrequired for VDE to react with violaxanthin but not with antheraxanthin.Otherwise, the detergent was necessary for the reaction on thelumenal surface. (Received September 5, 1997; Accepted October 19, 1997)