PURIFICATION AND PROPERTIES OF VIOLAXANTHIN DE-EPOXIDASE FROM SPINACH

The enzyme violaxanthin de-epoxidase (VDE) catalyzes the conversion of violaxanthin (V) to antheraxanthin (A) and zeaxanthin (Z). It has bee n purified 194-fold with a yield of 1.4% from a sonicate of thylakoids of Spinacea oleracea to a specific activity of 19 mu mol Z + A/min pe r mg protein. Purification steps included chromatography on DEAE-Sepha dex, hydrophobic interaction chromatography on Butyl Sepharose, isoele ctric-focusing, and gel filtration on Sephadex G-100. A single peptide band of 43.3 kDa was detected on SDS-PAGE, the apparent molecular mas s of native enzyme was 45.8 kDa by gel filtration, and the pI was 4.95 on isoelectric focusing. The enzyme required ascorbate for activity, had a pH optimum of 5.2 and was stimulated three-fold by the addition of monogalactosyldiacylglycerol (MGDG). The K(m)s for V and A were 5 a nd 5.3 mu M, respectively. VDE was inhibited 50% by dithiothreitol (DT T), mercaptoethanol, cysteine and o-phenanthroline at 0.055, 0.68, 2.7 and 0.025 mM, respectively. With both DTT and o-phenanthroline, the r ate of conversion of V to A was relatively unchanged whereas the conve rsion of A to Z was 50-80% inhibited. The enzyme also exhibited produc t inhibition by Z. (C) 1997 Elsevier Science Ireland Ltd.