Influence of lecithin liposomes on chlorophyllase-catalyzed chlorophyll hydrolysis: comparison of intramembraneous and solubilized Phaeodactylum chlorophyllase.

1.Chlorophyllase-catalyzed chlorophyll hydrolysis is greatly enhanced by the addition of divalent cations (Mg2+) combined with a reducing agent (dithiothreitol, ascorbate). A similar effect is obtained by the addition of lecithin. In the presence of lecithin, dithiothreitol has only slight or no influence on chlorophyll hydrolysis. Mg2+ eliminates the activating effect of lecithin. 2. In the absence of Mg2+ + dithiothreitol or of lecithin, Triton X-100 has a slight activating effect on chlorophyllase-catalyzed chlorophyll hydrolysis, but only at low concentrations (0.01--0.02%). In the presence of Mg2+ and dithiothreitol or of lecithin, Triton X-100 (greater than or equal to 0.02%) inhibits this reaction. 3. Whereas chlorophyllase combines with chlorophyll, no combination of chlorophyllase and lecithin could be detected. 4. Solubilized chlorophyllase is stabilized by its substrate, chlorophyll. Enzyme stabilization is eliminated by lecithin, whereas in the absence of chlorophyll, denaturation is somewhat increased by dithiothreitol. 5. No clear difference was found between the actions of intramembraneous and solubilized chlorophyllase. The results suggest that chlorophyllase is situated within membranes in such a way that the active group protrudes into the aqueous medium surrounding the membrane. 6. A hypothesis explaining the activating effects which Mg2+ combined with a reducing agent and lecithin have upon chlorophyllase-catalyzed chlorophyll hydrolysis is presented.