A NEW METHOD FOR THE DETERMINATION OF THE TRANSTHYLAKOID PH GRADIENT IN ISOLATED-CHLOROPLASTS - THE PH-DEPENDENT ACTIVITY OF VIOLAXANTHIN DE-EPOXIDASE

The activity of the enzyme violaxanthin de-epoxidase, VDase, in osmoti cally shocked chloroplasts from Spinacia oleracea cv. Monatol was empl oyed for the determination of the transthylakoid pH gradient, Delta pH . VDase activity was monitored as zeaxanthin formation by absorbance m easurements at 505 and 560 nm. For the calibration of VDase activity, the cation permeability of the membranes was increased with gramicidin D and formation of zeaxanthin was started by a pH jump towards acidif ication. The magnitude of the Delta pH determined this way, was indepe ndent of the intrathylakoid volume. The considerable independence of V Dase activity of the concentration of permeable cations indicated that VDase was an indicator of pH in the bulk aqueous phase of the thylako id lumen. ATP formation, assayed simultaneously with VDase activity, w as driven here by linear electron flow. The velocity of ATP formation, V-p, was linearly related to Delta pH. When Delta pH was adjusted by light, its relationship to V-p compared to that observed after a deter mination of Delta pH by C-14-methylamine uptake. When nigericin was em ployed for the adjustment of Delta pH, the relationships of Delta pH a nd V-p were different, depending on the method of Delta pH determinati on applied. The advantages and limitations of the new method are discu ssed.