Studies of crab digestive phospholipase acting on phospholipid monolayers: Activation by temperature
Résumé
The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. Using the classical emulsified system and the monomolecular film technique, we compared the interfacial properties of crab digestive phospholipase (CDPL) with those of the porcine pancreatic one (PPPL). A kinetic study on the surface pressure dependency of the two phospholipases was performed using monomolecular films of three substrates: di C12-PC (1.2-dilauroyl-sn-glycerol-3-phosphocholine); di C12-PG (1.2-dilauroyl-sn-glycerol phosphoglycerol) and di C12-PE (1.2- dilauroyl-sn-glycerol phosphoethanolamine). The use of a substrate in monofilm state allows the monitoring, during the biocatalysed reactions, of several physico-chemical parameters and permits the modification of the “quality of interface”. The effect of the temperature on the hydrolysis rate of these substrates was also checked. Our results show that specific activities of both phospholipases were affected by the variation of the subphase temperature. CDPL was irreversibly inactivated by p-bromo-phenacyl bromide, the specific inhibitor of secretory PLA2s. The hyperbolic behaviour observed was coherent with hopping mode of action, one of the two characteristic interfacial mechanisms of PLA2s.
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