Exploring Interfacial Hydrolysis Of Neutral Phospholipid Monolayer And Bilayer Catalyzed By Phospholipase C

Phospholipases,a category of enzymes hydrolyzing phospholipids,are widely distributed in all kinds of organisms.Besides the crucial roles in membrane homeostasis,phospholipases are also important to the digestion of nutrients and the formation of bioactive molecules.Phospholipase C（PLC）,an important type of phospholipases with the feature of hydrolyzing phospholipids at the position of the glycerophosphate bond,is not only directly involved in the metabolism of cell membranes,but also plays a key role in regulating multiple signaling pathways.PLC extracted from Bacillus cereus（BC-PLC）has been extensively studied owing to its similarity to hitherto poorly characterized mammalian analogues.This thesis focuses on investigating the interfacial hydrolysis mechanism of phosphatidylcholine（PC）monolayer and bilayer membranes catalyzed by BC-PLC by using sum frequency generation vibrational spectroscopy（SFG-VS）and laser scanning confocal microscopy（LSCM）.We found,upon interfacial hydrolysis,BC-PLC was adsorbed onto the lipid interface and catalyzed the lipolysis with no net orientation,as evidenced by the silent amide I band,indicating ordered PLC alignment was not a pre-requisite for the enzyme activity,which is very different from what we have reported for phospholipase A₁（PLA₁）and phospholipase A₂（PLA₂）（Phys.Chem.Chem.Phys.2018,20,63-67;Langmuir 2019,35,12831-12838;Langmuir 2020,36,2946-2953）.For the PC monolayer,one of the two hydrolysates,phosphocholine,desorbed from the interface into the aqueous phase;while the other one,diacylglycerol（DG）,stayed well packed with high order at the interface.For the PC bilayer,phosphocholine dispersed into the aqueous phase too,similar to the monolayer case;however,DG,presumably formed clusters with the unreacted lipid substrates and desorbed from the interface.With respect to both the monolayer and bilayer cases,mechanistic schematics were presented to illustrate the different interfacial hydrolysis processes.Therefore,this model experimental study in vitro provides significant molecular-level insights and contribute necessary knowledge to reveal the lipolysis kinetics with respect to PLC and lipid membranes with monolayer and bilayer structures.