Phospholipase C Hydrolysis of Phospholipids in Bilayers of Mixed Lipid Compositions

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Abstract
Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesicles. Phosphatidylcholine, phosphatidylethanolamine (also a substrate for the enzyme), sphingomyelin, and cholesterol have been mixed in various proportions, in binary, ternary, and quaternary mixtures. A lag period, followed by a burst of enzyme activity, has been found in all cases. The activity burst was always accompanied by an increase in turbidity of the vesicle suspension. Varying lipid compositions while keeping constant all the other parameters leads to a range of lag times extending over 2 orders of magnitude (from 0.13 to 38.0 min), and a similar variability is found in maximal enzyme rates (from 0.40 to 55.9 min-1). Meanwhile, the proportion of substrate that is hydrolyzed during the lag period remains relatively constant at 0.10% moles of total lipid, in agreement with the idea that enzyme activation is linked to vesicle aggregation through diacylglycerol-rich patches. Phosphatidylethanolamine and cholesterol enhance the enzyme activity in a dose-dependent way: they reduce the lag times and increase the maximal rates. The opposite is true of sphingomyelin. These lipids exert each its own peculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity can be to some extent predicted from its composition. Phospholipase C activity is not directly influenced by the formation of nonlamellar structures. However, the presence of lipids with a tendency to form nonlamellar phases, such as phosphatidylethanolamine or cholesterol, stimulates the enzyme even under conditions at which purely lamellar phases exist. Conversely sphingomyelin, a well-known stabilizer of the lamellar phase, inhibits the enzyme. Thus phospholipase C appears to be regulated by the overall geometry and composition of the bilayer.