The relation between the activity of a lecithinase and the electrophoretic charge of the substrate

Abstract

Aqueous emulsions of pure lecithin and lecithin containing various lipids (monophospho-inositide, cardiolipin), known to activate the hydrolysis of lecithin by a phospholipase B preparation, have been examined electrophoretically. Pure lecithin particles were electrophoretically net positive at the . optimum pH of the enzyme, whereas the activating lipids conferred on them a net negative surface. Other anionic amphipathic molecules (di-cetylphosphoric acid, sodium dodecyl sulfate, sodium hexadecyl sulfate, sodium l-palmitoyl-4-anisidine-2-sulfonate and sodium taurocholate) added to the lecithin were found to confer a net negative charge on its surface and also to activate the enzyme system. In a buffer of constant ionic strength, the enzymic activity commenced at a constant zeta potential, independent of the species of anionic amphipathic molecule used to activate the system. Inhibition of the enzyme system was produced by the addition to the bulk aqueous phase of various multivalent counter cations (Ca2+, UO2 2+, Th4+) and was complete when the observed zeta potential became zero. Addition of a cationic amphipathic molecule (cetyltrimethylammonium bromide) to the activated lecithin emulsion produced complete inhibition of the enzyme when the mobility of the emulsion particles fell below the critical activation value. Electrophore-sis of Penicillium notatum phospholipase B on paper and starch gel showed it was isoelectric at its pH optimum (3. 3). After starch-gel electrophoresis, lysolecithinase and lecithinase activity resided in the same protein band, which provides additional evidence that these enzymes are identical. It is concluded that a prerequisite for the attack of P. notatum phospholipase B on lecithin is that the substrate particles carry a minimum net negative zeta potential.