The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, we developed a mass-spectrometric (MS) assay and implemented it to study in detail the effect of acyl chain length and unsaturation of glycerophospholipids (GPLs) on their hydrolysis by three different secretory PLA2s (sPLA2s) from various sources. The key finding of this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis by these PLAs. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to be more important for substrate specificity. In the next study we used the MS assay developed in study I, to investigate whether substrate efflux propensity regulates the activity of the Ca2+ -independent PLA-Beta (iPLAβ). Our results strongly suggest that the activity of iPLAβ is also determined by the efflux of the GPL substrate from the membrane bilayer. Our last study was on the cytoplasmic PLA-alpha (cPLA2α) that has been implicated in the initiation of the inflammatory lipid-mediator cascade generating eicosanoids and platelet-activating factor. The study was carried out to understand to what extent accommodation in the catalytic site determines specificity for arachidonic acid (AA) and if efflux propensity plays a role in the substrate specificity of cPLA2α. Our results indicate that while accommodation of the substrate in the active site greatly contributes to the preferential hydrolysis of AA-containing GPLs by cPLA2α, efflux of the substrate from a membrane bilayer also plays a significant role. In summary, these studies not only provide information on the factors regulating the substrate specificity of various PLAs but also indicate that lateral arrangement of GPLs could be a key regulator of homeostatic PLAs like iPLAβ.The aim of this thesis was to study the substrate specificity of A-type phospholipases (PLAs) that belong to different sub-families in order to understand the key factors that regulate their activity. The experimental part of this thesis consists of three studies each one focusing on PLAs that belong to a specific subgroup. In the first study, a mass-spectrometric high-throughput assay was developed which allows us to monitor the hydrolysis of a multitude of GPL molecular species simultaneously. Specificities of three different sPLA2s were elucidated by this method. The key finding in this study was that efflux of the GPL substrate from the bilayer largely determines the rate of hydrolysis. In micelles, accommodation of the GPL acyl chains in the catalytic active site seems to contribute to substrate specificity. Our second study gives the first experimental evidence that efflux of the substrate from the bilayer is the rate-limiting factor in the hydrolysis of GPL molecules by iPLAβ. We observed that the activity of the enzyme correlates inversely with the hydrophobicity of the substrate. In the final study on cPLA2α, the key finding is that accommodation of the substrate molecule in the active site of the enzyme is crucial for the preferential hydrolysis of AA-containing GPLs along with efflux of the substrate from the bilayer whereas in the case of GPL substrate lacking AA, efflux from the bilayer seemed to be determing the rate of hydrolysis.
|Status||Publicerad - 8 jan 2016|
|MoE-publikationstyp||G5 Doktorsavhandling (artikel)|
- 1182 Biokemi, cell- och molekylärbiologi