Investigating porcine pancreatic phospholipase A2 action on vesicles and supported planar bilayers using a quartz crystal microbalance with dissipation.
We present an investigation of the activity of porcine pancreatic phospholipase A2 towards phospholipids. The phospholipids are presented in three different ways, namely as tethered vesicles, intact surface-bound vesicles, and supported planar bilayers (SPBs). The process is followed using a quartz crystal microbalance which measures both the frequency shift and the energy dissipation factor. This technique is very sensitive not only to the mass of the material deposited on the crystal, but also to its viscoelasticity. The breakdown of the phospholipid vesicles and bilayers consequently gives rise to very large signal changes. Enzyme binding is separated from vesicle hydrolysis using nonhydrolyzable ether lipids. Intact and tethered vesicles give rise to the same profile, indicating that direct immobilization of the vesicles does not affect hydrolysis significantly. The data fit well to a Voight-based model describing the change in film structure with time. Initial enzyme binding to intact vesicles is accompanied by a significant increase in layer thickness as well as a decrease in viscosity and shear modulus. This effect, which is less pronounced in SPBs, is probably mainly due to the accumulation of hydrolysis products in the vesicle prior to rupture of the vesicles and release of bound water, since it disappears when lysolipid is included in the vesicles prior to hydrolysis.
Department of Life Sciences, Aalborg University, Sohngaardsholmsvej 49, DK-9000 Aalborg, Denmark.
Pernille H
PH
Justesen
Tina
T
Kristensen
Tony
T
Ebdrup
Daniel
D
Otzen
D000818:Animals; D006868:Hydrolysis; D008051:Lipid Bilayers / Q000737:chemistry*; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry*; D054467:Phospholipases A2; D010743:Phospholipids / Q000737:chemistry*; D011791:Quartz; D013499:Surface Properties; D013552:Swine; D013997:Time Factors
D008051:Lipid Bilayers; D010743:Phospholipids; D011791:Quartz; D010741:Phospholipases A; D054467:Phospholipases A2