ON THE MECHANISM OF ACTION OF PHOSPHOLIPASE A

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Abstract
The phospholipase A of cobra (Naja naja) venom has been purified by density-gradient electrophoresis. The hydrolysis of lecithin and phosphatidylethanolamine by the purified enzyme has been studied in the aqueous system described by Magee and Thompson and correlated with the electrophoretic mobility of the substrate particles. Ca2+ ions are obligatory for the hydrolysis; they cannot be replaced by Mg2+ ions added to the buffer, nor by cetyltrimethylammonium hydroxide added to the lipid. The rate of hydrolysis of phosphatidylethanolamine increases as the pH is increased from 6 to 9-3; that of lecithin is comparatively unchanged. The hydrolysis of lecithin is inhibited by the addition of many anionic amphipathic molecules (e. g. dicetyl-phosphoric acid) to the substrate, but this is not related to the net negative potential produced on the lipid since the inhibition cannot be reversed when cetyltrimethylammonium hydroxide is added to restore the isoelectric state. Diethyl ether added to saturate the aqueous phase greatly stimulates the rate of hydrolysis of lecithin, whereas the breakdown of phosphatidylethanolamine is not affected. The addition of fatty acid to either lecithin or phosphatidylethanolamine inhibits the enzymic reaction.