The effect of phosphatidylinositols on cytosolic phospholipase A2 activity using a continuous fluorescent displacement assay.

Abstract

The 85 kDa, cytosolic phospholipase A, (cPLA, ) hydrolyses arachidonic acid from the sn-2 position of phospholipid substrates. Anionic phospholipids, particularly phosphatidylinositol-4,5bisphosphate (PIPJ, have been shown to enhance the activity of cPLA, [ 13. PIP, has become increasingly important because of its ability to specifically bind to many proteins, e.g. phospholipase C, via a Pleckstrin Homology domain (PH domain). Although the presence of a PH domain has not been reported in the case of cPLA,, the sensitivity of the enzyme to low molar concentrations of PIP, suggests the possibility of a specific interaction between the anionic phospholipid and cPLA,. In this study we have investigated the specificity of the effect of phosphoinositides on the activity of human cPLA,, using a continuous, fluorescence displacement assay. Small, unilamellar vesicles (SUVs) were prepared by mixing stock solutions of 1 -stearoyl-2-arachidonyl phosphatidylcholine (SAPC) and the appropriate amount of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PIP) or PIP,, up to 5mol%. The mixtures were dried under nitrogen, resuspended in 0.2M Tris/HCI, 0.2M NaC1, pH 8.0 and after gentle vortexing at room temperature, were subjected to probe sonication. The resulting solution of SUVs was added to assay buffer containing 0.1M Tris/HCI, 0.1M NaC1, pH 8.0, 300pM CaCI, and 1 I-(dansy1amino)undecanoic acid (DAUDA), to yield a final phospholipid concentration of l2.3nmoles/ml. The activity of cPLA, on the phospholipid vesicles prepared was measured using a rapid and continuous fluorescence displacement assay [2]. This assay utilises the interaction of DAUDA with fatty acid binding protein (FABP) to provide a high starting fluorescence, Hydrolysis of phospholipid substrate by CPLA, liberates fatty acids that displace DAUDA from FABP, resulting in a drop in fluorescence that is proportional to enzyme activity. The assay system is calibrated by the addition of known amounts of arachidonic acid. The effect of the incorporation of PI, PIP and PIP, into SAPC S W s , up to 5mol%, is shown in Fig. 1. The presence of PI appears to have no effect on the hydrolysis of SAPC by cPLA, at the molar ratios used. However incorporation of PIP and PIP, enhances this