Phospholipase C Hydrolysis of Phospholipids in Bilayers of Mixed Lipid Compositions

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.

A phosphatidylserine decarboxylase activity in root cells of oat (Avena sativa) is involved in altering membrane phospholipid composition during drought stress acclimation

“…An increased amount of PE is believed to influence the flexibility of the membrane and thereby facilitate invaginations during stress induced shrinkage. Besides affecting membrane structure, PE might also be important for incorporation and activity of certain enzymes [2,24] and possibly also has specific roles in cell signalling [4,29].…”

“…The integrity of membranes is vital both during the stress and, not least, during a subsequent rehydration, to maintain compartmentation and thereby functionality of the cell. The lipid composition is also important for a correct incorporation of membrane proteins and it is known that certain membrane integrated enzymes require the presence of specific lipids for full activity [2,24]. The plasma membranes of oat root cells are characterized by a high content of phospholipids [18].…”

Evidence for Contribution of Tripartite Hemolysin BL, Phosphatidylcholine-Preferring Phospholipase C, and Collagenase to Virulence of Bacillus cereus Endophthalmitis

“…These three phospholipids comprise about 86% of the mole percentage of phospholipids in the rabbit retina (43.9% PC, 34.7% PE, and 7.4% PS) (9). In addition, the presence of sphingomyelin (SM) in membranes inhibits the activity of PC-PLC (29); therefore, the exceptionally low concentration of SM in the rabbit retina (3.4%) most likely fosters the ability of PC-PLC to compromise retinal cell integrity. These factors are likely to be relevant to the human infection as well, since the phospholipid content of the human retina is 47.8% PC, 31.7% PE, 8.6% PS, and 4.3% SM (9).…”

Stimuli‐Responsive Lipid‐Based Self‐Assembled Systems

“…Liposomes have been designed to be activated by many macromolecules resulting in the collapse of the liposome or to induce membrane fusion under the action of the enzyme. Phospholipids are natural substrates for phospholipase enzymes -with phospholipase A2 (Andresen et al, 2005 ;Davidsen et al, 2001Davidsen et al, , 2003 and C (Montes et al, 2004 ;Nieva et al, 1989 ;Ruiz -Arguello et al, 1998 ) having been investigated for liposome destabilization. 9.2 ).…”