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-C-x-2-C-y-(COOH)-PC] and bolaform (1-C-x-2,2'-C-y-1'-C-x -PC) phosphatidylcholines were synthesized and examined as substrates for phospholipase A(2) (Naja naja naja) and C (Bacillus cereus). There is very little detectable phospholipase A(2) activity toward pure mic ellar 1-acyl-2-acyl-(omega-COOH) species. In addition, when these same omega-COOH species are present at concentrations above their CMCs, th ey are potent inhibitors of phospholipase A(2) hydrolysis of other mic ellar lipids. In contrast, phospholipase C hydrolysis of the same 1-ac yl-2-acyl-(omega-COOH)-PC species proceeds with rates comparable to th at of diheptanoyl-PC. The bolaform lipids, which are tethered through a common sn-2 acyl chain, (e.g., 1-C-8-2,2'-C-12-1'-C-8-PC) display qu ite different kinetic results. Under limiting Ca2+ conditions (100 mu M) all the available sn-2 acyl bonds of the dimer are hydrolyzed. Howe ver, at high Ca2+ concentrations (1-10 mM) the reaction curves have a biphasic nature, characterized by an initial burst of activity followe d by much slower rate. This is consistent with only the micellar 1-acy l-2-acyl-(omega-COOH)-PC produced in situ from phospholipase A(2) hydr olysis of the dimer acting as an inhibitor of subsequent phospholipase A(2) 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 te rms of the unique physical properties (as measured by NMR and fluoresc ence experiments) of these phospholipids.