The properties of a cloned human high-molecular-mass cytosolic phospholipase A2 investigated using a continuous fluorescence displacement assay: evidence for enzyme clustering on phospholipid vesicles.
The 85 kDa human cytosolic phospholipase A2 has been cloned and expressed 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 fluorescence 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 burst phase of hydrolysis represents the hydrolysis of these vesicles. This clustering produced a molar ratio of total phospholipid substrate to enzyme of about 450:1 at vesicle saturation with enzyme. Under limiting substrate conditions, the lower secondary rate that is observed results eventually in almost complete hydrolysis of the phospholipid; this was confirmed using radioactive substrate. Evidence is presented that during the initial burst phase, equivalent to hydrolysis of the outer monolayer of the vesicle, the enzyme remains tightly bound but is released as the reaction proceeds towards complete hydrolysis of the phospholipid substrate. In the presence of excess substrate, about 370 mol of fatty acid are released per mol of enzyme during the burst phase and it is calculated that this value also approximates to hydrolysis of the outer monolayer of the vesicle. It is proposed that the formation of a stable enzyme-vesicle complex during the burst phase of phospholipid hydrolysis may be due, at least in part, to protein-protein interactions between adjacent enzyme molecules in order to account for the clustering phenomenon.
Creaney
A
A
Department of Biochemistry, University of Southampton, U.K.
Masters
D J
DJ
Needham
M B
MB
Gordon
R D
RD
Mott
R
R
Wilton
D C
DC
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry; D013379:Substrate Specificity
D010743:Phospholipids; D010741:Phospholipases A; D054467:Phospholipases A2