Liposomes in the Study of Phospholipase A2 Activity
Introduction
Phospholipase A2 (PLA2) catalyzes hydrolysis of the sn-2 acyl chain of phospholipids. Physiologically, it appears to be involved in diverse functions such as digestion, membrane homeostasis, production of precursors for synthesis of several lipid mediators, defense against bacteria, clearing of dead or damaged cells, and as ligands for receptors.1, 2 Three basic types have been identified: secretory, cytosolic, and intracellular PLA2 (sPLA2, cPLA2, and iPLA2, respectively).3, 4 These three types differ in several ways including the compartment in which they act, their structure, and their dependence on calcium. Both cPLA2 and iPLA2 are large (40–85 kDa) intracellular enzymes; sPLA2 is a small (14 kDa) secretory enzyme that acts extracellularly. Secretory PLA2 requires calcium as a cofactor at micromolar to millimolar concentrations depending on the experimental conditions.5, 6 In contrast, activation of iPLA2 does not require calcium, and cPLA2 is regulated by calcium at the low concentrations applicable to the cytosol.3, 4 In addition, several isozymes of sPLA2 have been identified and are classified into various groups based on characteristic structural differences.2 This chapter focuses on methods that have been employed primarily with sPLA2. Nevertheless, many of them can be applied to studies of cPLA2.7, 8
The action of these enzymes toward liposomes has been studied for a variety of reasons. First, liposomes have provided a convenient in vitro reconstitution system for efforts to identify the basic enzymology of sPLA2. Second, liposomes have been useful in studies of potential inhibitors of the enzyme. More broadly, the sPLA2–liposome system has been used widely as a model for studying the fundamentals of lipid–protein interactions. This is true both from the perspective of basic studies to investigate the principles of catalysis at an aqueous–lipid interface as well as efforts to elucidate mechanisms by which physical properties of lipid aggregates influence the behavior of enzymes that bind reversibly to those aggregates.
In general, two steps are involved in the action of sPLA2 at interfaces (Fig. 1). In the first step, the enzyme adsorbs to the bilayer surface. The second step appears to be an activation step resulting in a productive complex between the bound enzyme and a phospholipid monomer from the membrane. Evidence from X-ray diffraction experiments and studies using polymerized phospholipids have suggested that for sPLA2 this second step involves physical migration of phospholipids upward from the plane of the bilayer into the enzyme active site.9, 10, 11 For sPLA2, the second step appears to be the one that requires calcium.12, 13, 14 Because the two steps are linked thermodynamically, the presence of calcium promotes the adsorption of the enzyme to the membrane surface.14
Section snippets
Choice of Experimental System
Both steps shown in Fig. 1 appear to be highly dependent on the physical properties of the membrane. This observation is the basis for much of the interest in studying sPLA2, but it has also led to considerable confusion in the interpretation of apparently conflicting results. The choice of the liposome system and the details of the reaction conditions, then, are especially important when conducting investigations with this enzyme. In fact, the physical properties of the membrane contribute
Liposomes
Procedures for producing the liposomes useful in studies of sPLA2 are generally well established and are described here only briefly. A few suggestions regarding issues of importance when working with an enzyme as sensitive to bilayer properties as is sPLA2 are included.
Assays of Hydrolysis
It is advisable to measure phospholipase activity over time rather than at a single point because the kinetics are rarely linear and are commonly complex. If a single point assay is desirable for screening a large number of compounds as potential inhibitors or for assaying column fractions during purification, it is recommended that either a charged or a micellar substrate be used. Several methods exist for assaying hydrolysis as a function of time. Here, we discuss indirect methods using pH
Assays of Binding
It is important for binding studies that the enzyme be inhibited. If hydrolysis were allowed to proceed, the high activity of the enzyme in cleaving bilayer phospholipids would quickly alter the bilayer composition and change the nature of the interface. There are several methods to inactivate the sPLA2: site-directed mutagenesis of active site residues, chemical modification of the active site histidine residue (e.g., 100 μM p-bromophenacyl bromide), the use of nonhydrolyzable substrates
Assays of Changes in Membrane Properties during Hydrolysis
One of the difficulties in studying sPLA2 is due to the change imposed on the substrate during sPLA2-dependent hydrolysis. As the hydrolysis products are formed, the membrane must accommodate both changes in volume of the membrane and the introduction of new species, lysophospholipid and fatty acid (depending on partition coefficients). One method to examine these membrane changes, and their effect of sPLA2, is through the introduction of membrane probes that are sensitive to subtle changes in
Fluorescence Imaging of Giant Unilamellar Vesicles
Giant unilamellar vesicles (GUVs) offer the researcher a model membrane system with several important features. GUVs have a minimum bilayer curvature in comparison with other commonly used liposome preparations, SUVs and LUVs, that may better model cellular membranes. A second, and more important, advantage of the GUV system is the ability to study a single vesicle. Bulk liposome studies generate information about a population of enzymes and vesicles, and calculations based on these data can
Concluding Remarks
Several of the techniques described here have also been applied to studies of the relationship between membrane structure and susceptibility to sPLA2 in human erythrocytes.84 Assays of hydrolysis by thin-layer chromatography or the fatty acid-binding proteins and the use of laurdan fluorescence and the two-photon microscopy technique have provided promising results. The simplicity of erythrocytes compared with nucleated cells and the considerable background knowledge available make erythrocytes
Acknowledgements
This work was supported by NSF Grant MCB 9904597 to Brigham Young University and by NIH Resource Grant RR03155 to the Laboratory for Fluorescence Dynamics. We appreciate the assistance of Rebekah Vest in preparing the manuscript.
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