Violaxanthin de-epoxidase.

Violaxanthin de-epoxidase catalyzes the de-epoxidation of violaxanthin to antheraxanthin and zeaxanthin in the xanthophyll cycle. Its activity is optimal at approximately pH 5.2 and requires ascorbate. In conjunction with the transthylakoid pH gradient, the formation of antheraxanthin and zeaxanthin reduces the photochemical efficiency of photosystem II by increasing the nonradiative (heat) dissipation of energy in the antennae. Previously, violaxanthin de-epoxidase had been partially purified. Here we report its purification from lettuce (Lactuca sativa var Romaine) to one major polypeptide fraction, detectable by two-dimensional isoelectic focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, using anion-exchange chromatography on Mono Q and a novel lipid-affinity precipitation step with monogalactosyldiacylglyceride. The association of violaxanthin de-epoxidase and monogalactosyldiacyglyceride at pH 5.2 is apparently specific, since little enzyme was precipitated by eight other lipids tested. Violaxanthin de-epoxidase has an isoelectric point of 5.4 and an apparent molecular mass of 43 kD. Partial amino acid sequences of the N terminus and tryptic fragments are reported. The peptide sequences are unique in the GenBank data base and suggest that violaxanthin de-epoxidase is nuclear encoded, similar to other chloroplast proteins localized in the lumen.

Effects of surfactants, pH, and certain cations on precipitation of proteins by tannins.

Tannic acid and pin oak tannins precipitate large amounts of the abundant leaf protein, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPC), over a wide pH range (6.15-9.30) in the presence of sodium, potassium, magnesium, and calcium ions at concentrations comparable to those reported in the gut fluids of lepidopteran herbivores. The presence of lysolecithin, a surfactant known to be present in the gut fluids of some insects, significantly reduces the amount of RuBPC precipitated under these conditions. We conclude that high detergency is far more effective than high alkalinity in countering the potential protein-precipitating properties of tannins. We further conclude that tannins do not deserve the status they were once accorded as general, all-purpose, dose-dependent, antidigestive defensive chemicals. We also describe the application of the Schaffner-Weissman protein assay for studying the protein-precipitating capacity of plant extracts. This method is far superior to the one we have used in our earlier studies.