Role of lateral phase separation in the modulation of phospholipase A2 activity.
Phospholipase A2-catalyzed hydrolysis of phosphatidylcholine large unilamellar vesicles is characterized by a period of slow hydrolysis followed by a rapid increase in the rate of hydrolysis. The temporal relationship between the burst of PLA2 activity and the lateral distribution of substrate and product lipids was examined by simultaneously recording product accumulation and the fluorescence of 1-pyrenyldecanoate, a fatty acid derivative sensitive to lipid distribution and lateral diffusion. The excimer: monomer ratio of the probe changes slowly prior to the burst in activity and then abruptly at the time of the burst. A partial phase diagram for the ternary codispersion of substrate and products (dipalmitoylphosphatidylcholine and 1:1 monopalmitoylphosphatidylcholine/palmitic acid) was constructed by differential scanning calorimetry and suggests gel/gel immiscibility in this system. Thus, the changes in pyrene fluorescence during the time course of hydrolysis appear to be due to lateral phase separation. The critical mole fraction of product both for lateral phase separation in the gel state and for elimination of the lag phase is approximately 0.083. The simultaneous recordings of PLA2 activity and pyrene fluorescence show that the lateral rearrangement of lipids begins prior to and continues during the rapid activation process of PLA2. Two possible effects of lateral phase separation are that concentration of the protein in the product-rich regions promotes putative dimerization or that formation of phase interface regions promotes enzyme activation.
Burack
W R
WR
Department of Biochemistry, University of Virginia Health Sciences Center, Charlottesville 22908.
Yuan
Q
Q
Biltonen
R L
RL
D004789:Enzyme Activation; D005453:Fluorescence; D006868:Hydrolysis; D007700:Kinetics; D008051:Lipid Bilayers; D010713:Phosphatidylcholines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010741:Phospholipases A; D054467:Phospholipases A2