Some Properties of Purified Phospholipase D and Especially the Effect of Amphipathic Substances

1. The soluble phospholipase D of cabbage was purified by heat treatment, acetone precipitation and electrophoresis on a density gradient of aqueous glycerol. 2. The purified enzyme slowly attacked a lecithin suspension whereas ultrasonically treated lecithin was hydrolysed more rapidly. 3. Diethyl ether stimulated the hydrolysis of both the lecithin suspension and ultrasonically treated lecithin. 4. Ca(2+) was essential for the hydrolysis (optimum about 0.04m); it could not be replaced by Mg(2+) or cationic amphipathic substances. 5. The reaction had a sharp pH optimum at pH5.4, irrespective of the physical form of the lecithin substrate or the activator used. 6. Anionic amphipathic substances such as dodecyl sulphate, phosphatidic acid, triphosphoinositide and monocetyl phosphoric acid, were potent activators of the reaction: other acidic lipids were relatively inactive. 7. Cationic amphipathic substances inhibited the hydrolysis; however, they also reversed the inhibition caused by using an excess of anionic amphipathic substance as activator. 8. The activation produced by amphipathic substances could not be correlated with their effect on the zeta-potential or size of the substrate particles. 9. The addition of activating anionic amphipaths to lecithin induces the latter to adsorb enzyme from solution. In the absence of Ca(2+) the enzyme is denatured on the highly negatively charged surface, but in the presence of Ca(2+) (or Mg(2+)) it is protected from denaturation. It is suggested that this adsorption is an essential prerequisite for ready enzymic hydrolysis. 10. The hydrolysis of lecithin by the enzyme was strongly inhibited by protamine sulphate (0.1mg./ml.) and by choline and ethanolamine. 11. Ultrasonically treated phosphatidylethanolamine, or mixed particles of phosphatidylethanolamine plus dodecyl sulphate, were slowly attacked by the enzyme provided that Ca(2+) was present.