Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A 2 (sPLA 2 ). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA 2 . Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A 2 was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required to induce susceptibility. Results from fluorescence spectroscopy and two-photon excitation scanning microscopy using the membrane probe laurdan argued that susceptibility to sPLA 2 is a consequence of increased order of membrane lipids.Under normal conditions, healthy cell membranes resist catalysis by secretory phospholipase A 2 (sPLA 2 ) 1 (1-4). However, they may become susceptible under circumstances that cause alteration of membrane physical properties (1-4). Previous studies using artificial membranes demonstrated that alterations that increase susceptibility generally increase the anionic charge of the outer leaflet, increase bilayer curvature, and/or decrease interactions among neighboring phospholipids (5-9). In some cases, enhanced susceptibility of artificial membranes depends on an increase in the order of the phospholipids (8, 10 -14). These changes increase susceptibility by augmenting the binding of sPLA 2 and/or by improving access of membrane phospholipids to the active site of the enzyme (5-12, 15, 16).It is not known whether the properties that induce susceptibility to sPLA 2 in artificial membranes also contribute to the vulnerability of biological membranes to attack by the enzyme. In order to address this issue, we manipulated various properties of erythrocyte membranes by preparing different types of ghosts as explained in the accompanying particle (17). We found that the factors that determined the degree of susceptibility were increased exposure of phosphatidylserine, an anionic phospholipid, and increased membrane order. These interpretations agreed with those from studies of susceptibility using artificial membranes (5-16). The next question, then, is whether these same factors are important in the hydrolysis of intact cells by sPLA 2 under conditions at which they have become susceptible such as in the presence...

Bilayers composed of phosphatidylcholine initially resist catalysis by phospholipase A2. However, after a latency period, they become susceptible when sufficient reaction products (lysolecithin and fatty acid) accumulate in the membrane. Temperature near the main bilayer phase transition and calcium concentration modulate the effectiveness of the reaction products. The purpose of this study was to examine the individual contributions of lysolecithin and palmitic acid to the susceptibility of dipalmitoylphosphatidylcholine vesicles and to rationalize the effects of temperature and calcium. Various fluorescent probes (Prodan, Laurdan, pyrene-labeled fatty acid, and dansyl-labeled phospholipid) were used to assess changes in the ability of the reaction products to perturb the bilayer and to affect the interactions with the enzyme. Un-ionized palmitic acid decreased bilayer polarity and perturbed the membrane surface exposing some of the Prodan to bulk water. Lysolecithin increased bilayer polarity and the rate of dipolar relaxation in response to the excited states of Laurdan and Prodan. A combination of the individual contributions of each product was observed when palmitic acid and lysolecithin were present together at low calcium, and the effects of lysolecithin dominated at high calcium. Palmitic acid, but not lysolecithin, promoted the binding of phospholipase A2 to the bilayer surface in the absence of calcium. Lysolecithin reduced the ability of fatty acid to enhance binding apparently by altering the structure of fatty acid domains in the membrane. Furthermore, increased temperature and ionization of the fatty acid tended to cause segregation of bound phospholipase A2 into domains poor in phospholipid content which presumably impeded bilayer hydrolysis. In contrast, un-ionized palmitic acid and lysolecithin promoted hydrolysis by augmenting a step distal to the adsorption of enzyme to the bilayer. This kinetic response to lysolecithin was calcium-dependent. A model accounting for these varied influences of the reaction products is presented.