PHOSPHATIDYLCHOLINE AS SUBSTRATE FOR HUMAN PANCREATIC PHOSPHOLIPASE-A(2) - IMPORTANCE OF THE PHYSICAL STATE OF THE SUBSTRATE

The long-chain phosphatidylcholine/sodium cholate aqueous system as su bstrate for human pancreatic phospholipase A2 (PLA2) was investigated. At a constant phosphatidylcholine (PC) concentration of 8 mM, the enz yme activity increased with a decrease in cholate (C) concentration up to a PC/C ratio of approximately 0.8 and then rather abruptly decreas ed to lower values at a ratio above 1.5. At ratios between 0.8 and 1.5 , an increasing lag phase in the PLA2 activity was seen, indicating a progressive decrease in substrate availability to the enzyme. Reaction mixtures with a PC/C ratio of up to 0.67 were optically clear solutio ns composed of mixed bile salt/PC micelles of increasing mixed micella r aggregate size. Ratios between 0.67 and 1.5 were characterized by an increase in turbidity (at 330 and 450 nm) due to increasing formation of vesicles or liposomes. Above a PC/C ratio of 1.5, a sharp increase in turbidity was seen due to increasing formation of bilayer structur es other than vesicles. Pure vesicles obtained by dialysis of mixed mi cellar solutions were not hydrolyzed by the enzyme. Addition of bile s alts reversed the inhibition which was accompanied by a decrease in tu rbidity. Phosphatidylcholine was preferred as substrate for human PLA2 when present in large mixed disc-like bile salt micelles. Vesicular o r other types of lamellar liquid-crystalline phases of long-chain phos phatidylcholine did not serve as substrate for PLA2.