The topographic evolution of PLA2-hydrolyzed DPPC monolayer depends on the mixing kinetics of substrate and products

LUISINA DE TULLIO, M. L. FANANI, BRUNO MAGGIO

Buzios, Rio de Janeiro Brasil

Congreso; VII Iberoamerican Congress of Biophysics; 2009

Sociedades Iberoamericanas de Biofisica

Phospholipase A2 is an interfacially activated enzyme with mM Ca2+ requirements that hydrolyses the sn-2 acyl ester linkage of glycerophospholipids. We have reproduced an experiment of Salesse’s group (BBA 1990. 1023:365-379) that showed by epifluorescence microscopy the topographic evolution of PLA2 hydrolyzed dipalmitoylphosphatidiylcholine (DPPC) monolayer at a lateral surface pressure allowing phase coexistence. The presence of nicks of liquid expanded phase at boundaries of liquid-condensed (LC) domains is an indirect evidence of enzymatic activity. It is generally conceived that lipolytic enzymes act at boundaries of different phases in physical contact with membrane defects. The objective of this work is to elucidate the role of PLA2 surface localization in the generation of monolayer topography. To prove that topographic changes are due to DPPC hydrolysis, the enzymatic activity was determined by measuring changes, as a function of time, of two independent parameters: surface potential and film area. We made enzyme-free premixed films of DPPC with the products of the reaction (Lyso-PC and palmitic acid) (1:1) in different proportions, and we did not found LC domains with nicks at the boundaries. In an attempt to reproduce a more similar situation of substrate-products mixing as when this is actively generated by the enzymatic reaction (far from equilibrium condition), we surface mixed a film of 1:1 products and a film of DPPC at 10mN/m. In the latter situation we could reproduce the nicks in LC domains. This suggests that enzyme localization at the domain boundary is not the only factor responsible for the topographic changes. As reported earlier for other system (J. Lipid Research 2008. 49:2347-2355) our results suggest that the kinetic of mixing-demixing among the different lipids are major factor responsible of the topographic evolution.