The activity of Triton X-100 soluble chlorophyllase in liposomes

Chlorophyllase (chlorophyll-chlorophyllidohydrolase, EC 3.1.1.14) was isolated and purified from Phaseolus vulgaris L. chloroplasts and etioplasts dissolved in 1% Triton X-100 and 10% glycerol. A 100 and 40-fold purification, respectively, was achieved. Enzyme preparations from both sources had similar affinities for chlorophyll a when assayed in a Triton X-100 medium. When electrophoresed in sodium dodecyl sulphate polyacrylamide gels the major band in both preparations migrated as a peptide of 30,000 daltons. Chlorophyll containing liposomes were also used as a substrate for chlorophyllase. The rate of hydrolysis did not follow Michaelis-Menten kinetics. When chlorophyllide a or methyl chlorophyllide a was incorporated in the liposomes, then in the presence of phytol dissolved in methanol, methylchlorophyllide a and chlorophyll a were shown to be synthesized. Apparently the purified enzyme in the presence of lipids, is endowed with both synthetic and hydrolytic activity.