Liquid-liquid immiscibility in model membranes activates secretory phospholipase A2

Secretory phospholipase A₂ (sPLA₂) hydrolyzes phosphatidylcholines (PC) within lipid bilayers to produce lyso-PC and a fatty acid, which can act as signaling molecule in biological membranes. The activity of sPLA₂ depends on the membrane structure. Bilayer defects, curvature, and gel-fluid micro-heterogeneity are known to activate sPLA₂. Here, we investigate if liquid-liquid immiscibility within model membranes is sufficient for sPLA₂ activation. The onset of the hydrolytic activity of cobra-venom sPLA₂ towards mixed monolayers of dimyristoyl-PC (DMPC)/cholesterol 2:1 (mol/mol) has been determined using infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated (PM-) IRRAS. The lag phase of sPLA₂ activity increases exponentially with rising surface pressures starting at 12 mN/m. This indicates that enzyme activation is hampered at higher surface pressures. Below 12 mN/m, no lag phase is observed, and sPLA₂ is efficiently activated. The surface pressure that is critical for sPLA₂ activation correlates with the critical miscibility pressure according to the phase diagram of DMPC and cholesterol. Thus, coexisting, liquid-phase domains provide sufficient boundaries to activate sPLA₂. Moreover, liquid-liquid immiscibility is an activating mechanism for sPLA₂ that also applies to biological membranes under physiological conditions because the corresponding bilayer structure is associated with that of membrane rafts.