THE PROPERTIES OF A CLONED HUMAN HIGH-MOLECULAR-MASS CYTOSOLIC PHOSPHOLIPASE A(2) INVESTIGATED USING A CONTINUOUS FLUORESCENCE DISPLACEMENTASSAY - EVIDENCE FOR ENZYME CLUSTERING ON PHOSPHOLIPID-VESICLES

The 85 kDa human cytosolic phospholipase A, has been cloned and expres sed in insect Sf21 cells, The pure enzyme has been investigated using a fluorescence displacement assay that provides a continuous record of phospholipid hydrolysis [Wilton (1990) Biochem. J. 266, 435-439]. The unusual kinetic properties of this enzyme, previously described using radioactive assays, were readily demonstrated using the continuous fl uorescence assay and were examined in detail. It is proposed that the enzyme clusters on the surface of a fixed number of substrate vesicles during the initial stages of catalysis and that the characteristic bu rst phase of hydrolysis represents the hydrolysis of these vesicles. T his clustering produced a molar ratio of total phospholipid substrate to enzyme of about 450:1 at vesicle saturation with enzyme. Under limi ting substrate conditions, the lower secondary rate that is observed r esults eventually in almost complete hydrolysis of the phospholipid; t his was confirmed using radioactive substrate. Evidence is presented t hat during the initial burst phase, equivalent to hydrolysis of the ou ter monolayer of the vesicle, the enzyme remains tightly bound but is released as the reaction proceeds towards complete hydrolysis of the p hospholipid substrate. In the presence of excess substrate, about 370 mol of fatty acid are released per mol of enzyme during the burst phas e and it is calculated that this value also approximates to hydrolysis of the outer monolayer of the vesicle. It is proposed that the format ion of a stable enzyme-vesicle complex during the burst phase of phosp holipid hydrolysis may be due, at least in part, to protein-protein in teractions between adjacent enzyme molecules in order to account for t he clustering phenomenon.