Micellar bolaform and omega-carboxylate phosphatidylcholines as substrates for phospholipases.

A series of mixed-chain diacyl-PCs which contain an omega-COOH on the sn-2 chain [1-Cx-2-Cy-(COOH)-PC] and bolaform (1-Cx-2,2'-Cy-1'-Cx-PC) phosphatidylcholines were synthesized and examined as substrates for phospholipase A2 (Naja naja naja) and C (Bacillus cereus). There is very little detectable phospholipase A2 activity toward pure micellar 1-acyl-2-acyl-(omega-COOH) species. In addition, when these same omega-COOH species are present at concentrations above their CMCs, they are potent inhibitors of phospholipase A2 hydrolysis of other micellar lipids. In contrast, phospholipase C hydrolysis of the same 1-acyl-2-acyl-omega-COOH)-PC species proceeds with rates comparable to that of diheptanoyl-PC. The bolaform lipids, which are tethered through a common sn-2 acyl chain, (e.g., 1-C8-2,2'-C12-1'-C8-PC) display quite different kinetic results. Under limiting Ca2+ conditions (100 microM) all the available sn-2 acyl bonds of the dimer are hydrolyzed. However, at high Ca2+ concentrations (1-10 mM) the reaction curves have a biphasic nature, characterized by an initial burst of activity followed by much slower rate. This is consistent with only the micellar 1-acyl-2-acyl-(omega-COOH)-PC produced in situ from phospholipase A2 hydrolysis of the dimer acting as an inhibitor of subsequent phospholipase A2 activity. Phospholipase C hydrolysis of the PC dimer and the sn-2 omega-COOH PC is rapid, with both available glycerophosphate groups cleaved at presumably the same rate. These results are discussed in terms of the unique physical properties (as measured by NMR and fluorescence experiments) of these phospholipids.