PHOSPHOLIPASE-C HYDROLYSIS OF PHOSPHOLIPIDS IN BILAYERS OF MIXED LIPID COMPOSITIONS

Phosphatidylcholine phospholipase C (EC 3.1.4.3) from Bacillus cereus has been assayed with substrates in the form of large unilamellar vesi cles. Phosphatidylcholine phosphatidylethanolamine (also a substrate f or the enzyme), sphingomyelin, and cholesterol have been mixed in vari ous proportions, in binary, ternary, and quaternary mixtures. A lag pe riod, followed by a burst of enzyme activity, has been found in all ca ses. The activity burst was always accompanied by an increase in turbi dity of the vesicle suspension. Varying lipid compositions while keepi ng constant all the other parameters leads to a range of lag times ext ending over 2 orders of magnitude (from 0.13 to 38.0 min), and a simil ar variability is found in maximal enzyme rates (from 0.40 to 55.9 min (-1)). Meanwhile, the proportion of substrate that is hydrolyzed durin g the lag period remains relatively constant at 0.10% moles of total l ipid, in agreement with the idea that enzyme activation is linked to v esicle aggregation through diacylglycerol-rich patches. Phosphatidylet hanolamine and cholesterol enhance the enzyme activity in a dose-depen dent way: they reduce the lag times and increase the maximal rates. Th e opposite is true of sphingomyelin. These lipids exert each its own p eculiar effect, positive or negative, either alone or in combination, so that the susceptibility of a given mixture to the enzyme activity c an be to some extent predicted from its composition. Phospholipase C a ctivity is not directly influenced by the formation of nonlamellar str uctures. However, the presence of lipids with a tendency to form nonla mellar phases, such as phosphatidylethanolamine or cholesterol, stimul ates the enzyme even under conditions at which purely lamellar phases exist. Conversely sphingomyelin, a well-known stabilizer of the lamell ar phase, inhibits the enzyme. Thus phospholipase C appears to be regu lated by the overall geometry and composition of the bilayer.