Characterization of Phospholipase A2
Our goal is to identify and characterize in vitro all of the individual phospholipases involved in a given cell’s regulation and to also characterize that regulation in the intact cell and tissue. To that end, our laboratory has focused initially on phospholipase A2 [Review: 163] where we obtained three distinct types of PLA in pure form and studied their in vitro activities. This includes several Ca2+-dependent secretory enzymes (sPLA2), the Ca2+-dependent cytosolic enzyme (cPLA2) and a Ca2+-independent PLA2 (iPLA2) [Review: 194] which we were the first to identify, purify and characterize (162) and study its inhibition (170). This Group VI enzyme has been cloned (192). We were the first to find that the Group V sPLA2 is expressed and secreted in response to stimuli (185) and we have identified critical residues in the Group IV cPLA2 (183) and have shown that it contains a binding site for phosphatidylinositol 4,5-bisphosphate (PIP2) which activates the enzyme (200+ 248).
Our goal is to identify and characterize in vitro all of the individual phospholipases involved in a given cell’s regulation and to also characterize that regulation in the intact cell and tissue. To that end, our laboratory has focused initially on phospholipase A2 [Review: 163] where we obtained three distinct types of PLA in pure form and studied their in vitro activities. This includes several Ca2+-dependent secretory enzymes (sPLA2), the Ca2+-dependent cytosolic enzyme (cPLA2) and a Ca2+-independent PLA2 (iPLA2) [Review: 194] which we were the first to identify, purify and characterize (162) and study its inhibition (170). This Group VI enzyme has been cloned (192). We were the first to find that the Group V sPLA2 is expressed and secreted in response to stimuli (185) and we have identified critical residues in the Group IV cPLA2 (183) and have shown that it contains a binding site for phosphatidylinositol 4,5-bisphosphate (PIP2) which activates the enzyme (200+ 248).
The three enzymes mentioned are the main examples of what is a growing superfamily of phospholipase A2's [Review: 233]. For the in vitro study of these enzymes, we have cloned and expressed human and murine examples of each type of PLA2 using yeast Pichia pastoris, bacterial E. coli and the Baculovirus/Sf9 cell based expression systems and even chemical synthesis (209). We are currently carrying out expression studies, site-directed mutagenesis, kinetic analysis, and NMR and mass spectrometric studies of substrate and inhibitor interactions.
The three enzymes mentioned are the main examples of what is a growing superfamily of phospholipase A2's [Review: 233]. For the in vitro study of these enzymes, we have cloned and expressed human and murine examples of each type of PLA2 using yeast Pichia pastoris, bacterial E. coli and the Baculovirus/Sf9 cell based expression systems and even chemical synthesis (209). We are currently carrying out expression studies, site-directed mutagenesis, kinetic analysis, and NMR and mass spectrometric studies of substrate and inhibitor interactions.