Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase.

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4 -yl) amino]-caproyl] phosphatidylcholine (C6-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A2 (PA2) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C6-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca++ was required for PA2 catalysis. Identification of the products of hydrolysis showed cleavage at the sn-1 and sn-2 positions for LpL and PA2, respectively. For PA2, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.