Pharmac. Ther.Vol. 54, pp. 26%295, 1992 Printed in Great Britain. All rightsreserved

0163-7258/92 $15.00 © 1992PergamonPressLtd

Associate Editor: A. L. HARVEY

MEMBRANE STRUCTURE, TOXINS AND PHOSPHOLIPASE A 2 ACTIVITY LEO P. VERNON* and JO~IN D. BELL~" Departments of*Chemistry and t Zoology, Brigham Young University, Provo, UT 84602, U.S.A. Abstraet--The phospholipid-hydrolyzing enzyme phospholipase A 2 (PLA2) (EC exists in several forms which can be located in the cytosol or on cellular membranes. We review briefly cellular regulatory mechanisms involving covalent modification by protein kinase C and the action of Ca 2÷ , cytokines, G proteins and other cellular proteins. The major focus is the role of phospholipid structure on PLA2 activity, including (1) the mechanism of PLA2 action on synthetic phospholipid bilayers, (2) perturbation of synthetic and cellular membranes with lipophilic agents and membrane-interactive peptides and (3) the ability of these agents to activate endogenous PLA2 activity, with emphasis on the venom and plant toxins melittin, cardiotoxin and Pyrularia thionin.

CONTENTS 1. Introduction 2. Structure of Phospholipase A2 (PLA2) 3. Activation of Cellular PLA 2 3.1. Ca z+ 3.2. Covalent modification 3.3. G proteins 3.4. Other peptides 3.5. Cytokines 4. Role of Membrane Structure in PLA2 Activation 4.1. Cellular membranes 4.1.1. Membrane perturbation by nonprotein agents 4.1.2. Membrane perturbation by proteins and peptides 4.2. PLA 2 activation by plant and venom toxins 4.2.1. Melittin 4.2.2. Cardiotoxin 4.2.3. Pyrularia thionin 4.3. Models of PLA 2 activation with synthetic bilayers 4.3.1. General hypothesis 4.3.2. Binding model 4.3.3. Monomer models 4.3.4. Dimer models 5. Summary References

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1. I N T R O D U C T I O N Phospholipase A2 (PLA2) (EC catalyzes the hydrolysis of phospholipid molecules at the sn-2 position to create free fatty acids and 1-acyllysophospholipids. The enzyme has been studied extensively during the past few decades because of its physiological and pathological importance Abbreviations: CL, cardiolipin; CTX, cardiotoxin; DAG, diacylglycerol; DMPC, dimyristoylphosphatidylcholine; DPPA, dipalmitoylphosphatidic acid; ILl, interleukin 1; PA, phosphatidic acid; p-BPB, p-bromophenacyl bromide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PGE2, prostaglandin Ez; PI, phosphatidylinositol; PLA2, phospholipase As; PS, phosphatidylserine; P. thionin, Pyrularia thionin; Ira, transition temperature; TNF, tumor necrosis factor. 269



and also because of the interesting biochemical and biophysical properties associated with it (reviewed in Verheij et al., 1981; Jain and Berg, 1989). Physiologically, the products of the reaction catalyzed by PLA2 are themselves precursors for other hormones (prostaglandins, leukotrienes, platelet-activating factor) as well as possible intracellular second messengers (Dreher and Hanley, 1988; Oishi et al., 1988). In addition, PLA2 appears to fulfill an important role in maintaining the composition and health of cell membranes (Sevanian et al., 1988). Pathologically, excess PLA: activity has been implicated in a number of disease states including trauma, viral infection, shock, inflammatory disease and pancreatitis (see Chang et aL, 1987; Scheuer, 1989 for a more comprehensive treatment of this subject). Obviously, the activity of an enzyme involved in so many processes must be regulated precisely by living cells and organisms. The scope of this review is to present some of what is known concerning the possible mechanisms by which the activity of PLA 2 is regulated acutely in the cell. Specifically, we focus on the role that membrane structure and dynamics may have in modulating the activity of the enzyme and how certain peptide molecules may participate in that process. 2. STRUCTURE OF PHOSPHOLIPASE A 2 (PLA:) Phospholipase A: (PLA2) has been purified from a wide range of sources. The PLA2 that have been studied most intensely are small soluble enzymes of MW around 14,000 (Verheij et aL, 1981). These enzymes are relatively simple and inexpensive to purify in large quantities and may be obtained from the mammalian pancreas and from the venom of snakes. Many have been sequenced completely and three dimensional structures have been obtained using X-ray crystallography for several (Verheij et al., 1981; Renetseder et al., 1985; Holland et al., 1990; White et aL, 1990). Furthermore, these enzymes are very stable, partly because of the seven disulfide bridges common to these proteins. In general, they may be assigned to one of two major groups based on differences in the primary sequence and the position of one of the disulfide bridges (Maraganore and Heinrikson, 1986). Pancreatic PLA2 and the enzyme from the Elapidae and Hydrophidae families of snakes belong to group I. The other snake venom enzymes generally belong to group II. Many of the PLA 2 that have been isolated from cellular sources bear strong sequence homology to the pancreatic and snake venom enzymes. For example, PLA2 resembling group I enzymes have been isolated from rat spleen, gastric mucosa and lung (Tojo et al., 1988; Sakata et al., 1989). Group II PLA2 have been found in rat and human platelets, human rheumatoid arthritic synovial fluid, rat liver and rat and human spleen (Hayakawa et al., 1988; Kramer et al., 1989; Seilhamer et al., 1989; Aarsman et aL, 1989; Ono et al., 1988; Kanda et al., 1989). The three-dimensional structure of the human synovial fluid enzyme has been determined from X-ray crystallography data (Wery et al., 1991; Scott et al., 1991) and appears very similar to the structures of the venom enzymes. Therefore, one assumes that the profusion of data attempting to address the mechanism of PLA2 activation using the pancreatic and venom enzymes should be directly relevant to these relatively scarce enzymes present in mammalian cells. The system appears not to be so simple, however. These small PLA2 isolated from the tissues described above appear frequently, if not always, to be secretory enzymes that may be released from cells under certain conditions or which may be contained in lysosomes. Recent work has demonstrated that the family of PLA 2 may be both much larger and more heterogeneous than originally thought. For example, larger PLA 2 have been isolated from a macrophage cell line (70 kDa), guinea pig intestine (97 kDa), canine myocardium (40 kDa), a human monocyte cell line (ll0 kDa), rabbit platelets (88 kDa) and human platelets (90 kDa) (Leslie et al., 1988; Gassama-Diagne et al., 1989; Hazen et al., 1990; Clark et al., 1990; Kim et al., 1990; Takayama et al., 1991). Which, if any, of these enzymes are the ones regulated physiologically by hormone receptors remains unresolved. It does appear clear, however, that the reaction products generated by either the small or large PLA2 may function as precursors for the prostaglandins, leukotrienes and platelet activating factor (Chang et al., 1987; Leslie et al., 1988; Kim et al., 1990; Takayama et aL, 1991). Therefore, it would seem important that the body is capable of regulating the activity of all PLA:.

Phospholipase A 2 activity


3. ACTIVATION OF CELLULAR PLA2 The two general possibilities for activation of PLA2 in the cell are modulation of either the enzyme and/or the phospholipid bilayer substrate. The first involves a modification of the protein structure to either increase the extent of the initial binding of the enzyme to a site on the membrane or to facilitate the subsequent enzymatic activity of the bound enzyme through a conformational change in the protein. The second is similar, but involves changes in the phospholipid bilayer that could either facilitate enzyme binding or result in a bilayer configuration more conducive to high enzyme activity. In this review we will briefly consider first those agents which activate PLA2 by modifying the protein. The majority of the review will then focus on conditions and agents which modify the bilayer structure of synthetic and cellular membranes to affect PLA2 activity. 3.1. Ca 2÷ In general, Ca 2+ is required for PLA 2 activity and there is a high affinity site near the catalytic site (Verheij et al., 1981; Jain and Berg, 1989). Therefore, an obvious mechanism by which hormones and other factors could activate PLA2 acutely would be through an increase in intraceUular Ca 2+ concentration. Indeed, several examples of such regulation have been reported (Slivka and Insel, 1987; Brooks et al., 1989; Channon and Leslie, 1990), including the use of the ionophore A23187 to facilitate Ca 2+ entry and thus increase cellular PLA 2 activity (Shier et al., 1982; Chakraborti et al., 1991). Nevertheless, some evidence suggests that increases in intracellular Ca 2+ may not always be required (Crouch and Lapetina, 1988; Pierik et aL, 1988) and PLA 2 in organelles such as the lysosome are CaE+-independent (Kawaguchi and Shier, 1987). As shown below, intracellular Ca: ÷ flux is clearly one, but not the only regulating factor in the cell. Some reports suggest that PLA2 may be partly regulated by intracellular pH. Specifically, an acute increase in intracellular pH has been correlated with enhancement of PLA2 activity (Baron and Limbird, 1988; Sweatt et al., 1986). 3.2. COVALENT MODIFICATION

Covalent modification by phosphorylation of PLA 2 is another proposed regulatory mechanism that has been corroborated by experimental data. In general, it appears that protein kinase C may directly or indirectly activate intracellular PLA2 via protein phosphorylation (Whatley et al., 1989; Halenda et al., 1989; Zor et al., 1990; Gronich et al., 1988; Kanterman et al., 1990; Weiss and Insel, 1991; Calignano et aL, 1991). In several reports, the activity of protein kinase C in the cell was correlated with the action of specific hormones or agents such as arginine vasopressin, dopamine, bradykinin, epinephrine and phorbol ester, all of which activate PLA 2 (Gronich et aL, 1988; Kanterman et al., 1990; Weiss and Insel, 1991; Parker et al., 1987). Since protein kinase C is activated by diacylglycerol (DAG), a product of phospholipase C activity, one needs also to consider this hydrolytic enzyme in the regulation of PLA2 activity. Some agents appear to act through more than one mechanism, since bradykinin and epinephrine also involve the activation of G proteins, as discussed below. It has also been suggested that tyrosine kinase activity of the epidermal growth factor receptor is involved in regulating PLA2 activity (Goldberg et al., 1990). Platelet-derived growth factor also stimulates growth of 3T3 cells by activation of an endogenous PLA2 (Shier and Durkin, 1982). Novel activation mechanisms, involving acylation of specific lysine residues in PLA 2 by fatty acid, or cross-linking of specific lysine and glutamine residues by a transglutaminase, have also been proposed (Cordella-Miele et al., 1990; Cho et al., 1988; Tomasselli et al., 1989). 3.3. G PROTEINS PLA2 activity in cells is stimulated by a number of hormones whose binding to a receptor on the cell membrane is coupled to the activation of G proteins. PLA 2 activation in this manner resembles the activation of adenylate cyclase by similar GTP-binding transducer proteins. The mechanism whereby these G proteins activate endogenous PLA2 enzymes is not known, but there is evidence that PLA2 no longer requires Ca 2÷ after being activated by a receptor (Bormann et al.,



1984; Bicknell and Valee, 1989). It appears there are multiple G proteins linked to PLA2 activation, but none has been completely characterized. Originally, it was thought that the fl? subunits of the G proteins were the molecular entities responsible for stimulation of PLA 2 (Jelsema and Axelrod, 1987). While such may be true, there is evidence that under certain conditions the ct subunit is involved (Gupta et al., 1990). Cholera toxin stimulates Gs~t, but can stimulate (Burch et al., 1988b) or inhibit (Jelsema, 1987) PLA2 depending on the receptor or cell type. Pertussis toxin inhibits G~ct (Gilman, 1987) and transducin (Stryer, 1986), but can inhibit or stimulate PLA2 (Burch et al., 1988b). It is beyond the scope of this review to consider the role of G proteins in more detail and the reader is referred to two reviews by Burch (1989, 1990). Information concerning what little is known about the reaction mechanism for the various G proteins involved is discussed and the former review lists with references the various tissues involved, agonists tested and toxin sensitivity. The agonists include ~radrenoceptor agonists, bradykinin, thrombin, ras gene product, fMetLeuPhe, GTP, light, G protein fl? subunit, IgG, lipopolysaccharide, FRMFamide, leukotrienes C4 and D4 and tumor necrosis factor. Later publications show that stimulations by angiogenin (Bicknell and Valee, 1989) and IgE (Narasimhan et al., 1990) also involve G proteins. 3.4. OTHER PEPTIDES

Other peptides may also regulate PLA2 activity directly. It had been suggested for many years that the enzyme might be inhibited by the presence of specific membrane-binding proteins called lipocortins. However, recent evidence suggests that the effect of lipocortins, if physiologically relevant, probably does not involve direct interactions between the polypeptide and PLA: (Davidson et al., 1986). Another peptide that has been proposed to regulate PLA2 activity directly has been isolated from murine smooth muscle, bovine endothelial cells and human arthritic synovial fluid (Clark et al., 1987; Bomalaski et al., 1989). The peptide bears structural similarity to the bee venom PLA2-stimulating peptide melittin and is recognized by antibodies prepared against the latter (Clark et al., 1987). Interestingly, melittin is thought to activate PLA 2 by perturbing the bilayer rather than through a direct interaction with the enzyme (Conricode and Ochs, 1989). One can speculate that the mechanism of action for this new stimulatory peptide could be similar to that of melittin. An apparently distinct PLA2-stimulating peptide has been identified in murine embryonic genital tracts (Gupta and Braun, 1990). 3.5. CYTOKINES

Since prostaglandins are produced from arachidonic acid and are important in inflammation reactions, the role of the cytokines interleukin 1 (ILl) and tumor necrosis factor (TNF) in stimulating the production of prostaglandin E 2 (PGE2) has been studied extensively. ILl induces PLA 2 activity in rabbit chondrocytes (Gilman et al., 1987) and PGE2 production in fibroblasts (Zucali et al., 1986), pancreatic islets (Hughes et al., 1989), 3T3 fibroblasts (Burch et al., 1988a) and fetal mouse bones (Sato et al., 1986). Stimulation of PLA2 activity by TNF has been shown in endothelial cells (Clark et al., 1988), tumor cell lines (Neale et al., 1988) and resting macrophages (Bachwich et al., 1986). Combinations of ILl and TNF have been studied with fibroblasts (Elias et al., 1987), human synovial cells (O'Neill and Lewis, 1989) and human glomerular mesangial cells (Topley et al., 1989). A stimulation of PGE2 production could involve stimulation of existing PLA 2 or increased synthesis of new PLA2 or cyclooxygenase. ILl and TNF stimulate the synthesis and secretion of extracellular PLA2 from rat mesangial cells (Schalkwijk et al., 1991) and from rat bone-forming cells (Vadas et al., 1991). ILl was shown to increase PLA2 activity in rabbit chondrocytes (Chang et al., 1986) and Kerr et al. (1989) demonstrated that the mechanism could involve an increase in messenger RNA for PLA2. Increases in PLA: messenger RNA were also observed with mesangial cells treated with ILl (Nakazato et al., 1991) and with human hepatoma cells treated with ILl, TNF and interleukin 6 (Crowl et al., 1991). In the latter case, interleukin 6 was the most potent stimulant. Enhanced PGE2 production in human fibroblasts by ILl was blocked by addition of actinomycin D or cycloheximide (Newton and Covington, 1987) and the level of cyclo-oxygenase mRNA was markedly enhanced in human endothelial cells after treatment with ILl (Maier et al., 1990). The effect of ILl on bradykinin receptor-mediated generation of

Phospholipase A 2 activity


PGE 2 was studied with Swiss 3T3 cells. Pretreatment with ILl induced synthesis of PLA 2 and cyclo-oxygenase de novo, but not phospholipase C or PGE isomerase (Axelrod, 1990). Consequently, ILl and TNF do not appear to be involved in activation of existing PLA2 molecules in the cell, but instead stimulate enzyme synthesis. The data reviewed above focus on major known regulatory mechanisms in the cell. Another important method of regulating PLA2 activity, modulation of the phospholipid bilayer structure, needs to be considered. As discussed above, DAG is produced by the action of phospholipase C and it affects PLA2 activity by stimulating protein kinase C. DAG may also function by altering the physical properties of the membrane. Indeed, some investigators feel that modification of phospholipid bilayer properties may play a large role in the regulation of PLA2 activity by hormones and other cellular processes (Achari et al., 1987; Gheriani-Gruska et al., 1988; Pieterson et al., 1974). We agree with this concept and will present supporting evidence below.

4. ROLE OF MEMBRANE STRUCTURE IN PLA2 ACTIVATION The importance of membrane structure on PLA 2 activity became apparent when it was discovered that the enzyme is much more active toward phospholipid organized in micelles or monolayers than toward monomers of the lipid (Pieterson et al., 1974; Verger et al., 1973). This phenomenon was termed 'interracial activation' and much effort has been channeled into understanding the molecular basis of the effect (Verheij et al., 1981). One possibility is that the conformation of the enzyme is different in the presence of aggregated phospholipid than with monomeric substrate (Verger et al., 1973). Another distinct, but not exclusive, possibility is that the conformation of the phospholipids is more favorable for hydrolysis when organized into a lipid aggregate (Barlow et al., 1988). It was also recognized early that the activation of PLA 2 in the presence of aggregated phospholipid is frequently time-dependent. Lag times prior to full activity have been demonstrated with both monolayers and bilayers as substrates for the enzyme (Verger et al., 1973; Apitz-Castro et al., 1982; Bell and Biltonen, 1989). In the case of large vesicles of saturated phosphatidylcholines (PC), the increase in activity at the end of the lag phase can be very abrupt (e.g. 100-fold increase in activity within a few seconds after a lag time of over an hour) (Bell and Biltonen, 1989). With some PLA2, the tryptophan fluorescence also increases abruptly with the activity (Bell and Biltonen, 1989). Whether the apparent activation that occurs with aggregation of the phospholipid substrate is the same process that occurs during time-dependent activation is not known. Nevertheless, both the activity of the enzyme toward aggregated phospholipid and the length of the lag phase are extremely sensitive to perturbation of the physical properties of the bilayer. Phospholipid bilayers undergo physical transitions from an ordered gel phase to a disordered liquid crystalline phase at a specific temperature (tm) depending on the composition of the fatty acid chains and the nature of the polar head-group. Both the rate of hydrolysis of phospholipid and the length of the lag time depend on the experimental temperature relative to tm (Bell and Biltonen, 1989; Op Den Kamp et al., 1974; Menashe et al., 1981). In general, the optimum temperature for the apparent activation of the enzyme is at or near the tin. The length of the lag phase in monolayers is also influenced by the lateral pressure of the monolayer (Verger et al., 1973). In analogy to the effect of temperature, the lag phase is shortest at a specific pressure and increases either at pressures above or below the optimal pressure. PLA2 is also sensitive to the size of the phospholipid aggregate. For example, micelles and small unilamellar vesicles (in the gel phase) of zwitterionic phospholipid are hydrolyzed instantaneously by the enzyme (Verheij et al., 1981; Menashe et al., 1986; Wilschut et al., 1978). As the size of vesicles increases and the bilayer curvature therefore decreases, lag phases begin to occur (Gheriani-Gruszka et al., 1988; Wilschut et al., 1978). Osmotic shock of large vesicles which introduces curvature into the near-planar bilayer reduces the lag time to near zero (Verheij et al., 1981; Lichtenberg et al., 1986). Most of the studies that have elucidated the details of these phenomena have used either pancreatic or snake venom enzymes. However, it appears that cellular PLA2 also exhibit similar dependence of their activity on the physical properties of the membrane



(Kannagi and Koizumi, 1979; Petkova et al., 1987; Lenting et al., 1988; Leslie and Channon, 1990; Malis et al., 1990). The sensitivity of PLA 2 to the physical properties of the bilayer suggests the possibility that such could be exploited by cells as an additional means of regulating the activity of the enzyme. 4.1. CELLULARMEMBRANES Cellular membranes are complex and delicately balanced structures which regulate diffusion to allow compartmentalization of cellular components, transport selected compounds into and out of the cell, transmit hormonal and other extracellular signals into the cell via second messengers, serve as the matrix for many enzymes and act as energy transducers to produce ATP. The complexity of the cellular membrane is expressed by the heterogeneous phospholipid content, the wide variety of integral proteins incorporated into the bilayer and the many peripheral proteins bound to the membrane surface. The complexity of the responses of the phospholipid bilayer to external agents is related to the properties of the different individual phospholipids with their varying headgroup size, electrostatic charge and polarity as well as the length and degree of unsaturation of the acyl chains. Other differences exist in the content of sphingolipids and cholesterol, as well as the asymmetric distribution of the membrane components on the two leaflets of the bilayer. The cell membrane interacts with its environment by the integral proteins embedded in and the peripheral proteins bound to the membrane surface and also by the exposed phospholipid areas of the membrane. There is ample evidence to show that the phospholipids, sphingolipids and cholesterol are not distributed homogeneously throughout the membrane and segregation of phospholipids occurs in the bilayer to form specific phospholipid domains (Curtain et al., 1988). Nonbilayer structures are also formed under some conditions, initiated by phosphatidylethanolamine (PE) and hydrolytic products of phospholipids. The integral proteins also bind certain lipids preferentially. All of these heterogeneities result in protein-lipid and lipid-lipid interfaces that have nonbilayer phospholipid structures, which can serve as the locus of local interactions with external proteins. Agents which modify the membrane structure would be expected to modify enzymatic reactions which take place on the membrane surface, especially those of PLA> The membrane modifications to consider are the following. (1) Change in membrane fluidity. This is a general term which is best described as simply the inverse of membrane viscosity, but can be measured by several operational parameters (Shinitzky, 1984). Increasing membrane fluidity increases the lateral and rotational movement of membrane components, resulting in a more flexible structure which could facilitate PLA 2 reaction with membrane phospholipids. (2) Formation of specific phospholipid domains. One common feature among various types of membrane perturbations that influence PLA2 activation is the existence of domains of one state or type of lipid within the bilayer. For example, it has been postulated that domains of gel and liquid crystalline lipid coexist at tm and this is related to the activation of PLA2 (Lichtenberg et al., 1986). Novel evidence for that proposal was obtained by attempts to image directly the presence of domains at t m by fluorescence microscopy and correlate the hydrolysis of the lipid with the existence of such domains (Grainger et al., 1989). (3) Nonbilayer structures. The formation of nonbilayer structures, such as the hexagonal II (Hu) structure, results in relatively disorganized interfaces which could facilitate enzymatic attack. (4) Other physical properties. Physical properties such as surface pressure and membrane potential could also affect enzyme binding and activity. (5) Phospholipid asymmetry. Phospholipid composition and distribution in the two membrane leaflets, especially the acidic phospholipids, affects the surface properties of the membrane. 4.1.1. Membrane Perturbation by Nonprotein Agents

Because of the hydrophobic nature of the phospholipid bilayer core, it is expected that any lipophilic molecule will affect the polymorphic nature of membrane bilayers in some fashion and

Phospholipase A 2 activity


major changes can be translated into altered cellular responses. For example, dibucaine and chlorpromazine induce H . structures in synthetic membranes containing unsaturated cardiolipin (CL) and phosphatidic acid (PA) (Cullis et al., 1978; Verkleij et al., 1982) and whereas ethanol and butanol stabilize the bilayer form, n-alcohols of chain length of 6 or longer as well as alkanes promote the formation of H . structures in egg PE systems (Cullis et al., 1980; Hornby and Cullis, 1981). The effect of simple organic compounds such as aliphatic alcohols, acids and amines upon phospholipid order in bilayers can be demonstrated by measuring the transition temperature (tin) for conversion of the bilayer from the gel to the liquid crystal state. With synthetic bilayers of dipalmitoylphosphatidylcholine,n-alcohols of 10 or fewer carbons lower the transition temperature and those of 12 or above increase it (Eliasz et al., 1976). With dimyristoylphosphatidylcholine (DMPC) bilayers, the changes occur between alcohols of 8 and 10 carbons (Lee, 1976). Experiments utilizing various biophysical techniques with pure and mixed lipid dispersions and aliphatic alcohols show that the short chain alcohols make the bilayer more fluid and depress the tm, whereas the long-chain alcohols elevate tm and decrease fluidity (Pringle and Miller, 1979; Jain and Wu, 1977; Jain et al., 1978; Hui and Barton, 1973). Alkanes and alkanols from 6 to 12 carbons induce the nonbilayer H u phase in egg yolk PE membranes (Hornby and Cullis, 1981). A systematic study of the effect of n-alcohols on soluble PLA2 activity with synthetic vesicular membranes of PC involved the use of 3~p_ and ~H-NMR to show that the initial rate of hydrolysis increased after adding n-alcohols in a chain-length manner, peaking at 8 carbons (Kaszuba and Hunt, 1990). The rate of hydrolysis correlated with the degree of clustering of the n-alcohols in the membrane. Shinitzky (1984) discusses and reports on the effect of membrane fluidity on the activity of membrane enzymes. Changing the fluidity of cellular membranes by variation of phospholipid and cholesterol content increases or decreases the activity of membrane-related enzymes. Experiments have been performed in one of our laboratories (L.V.) on the effect of long chain alcohols and glycols (8 to 14 carbons) on the induction of PLA 2 activity by following the release of 3H-arachidonic acid from loaded Swiss 3T3 cells (Evans, 1990). With n-alcohols, peak activity occurred at 12 carbons. Labeled arachidonic acid from cells exposed to 8 and 14 carbon alcohols at 300/.tM was very slight. Similar data were obtained with a series of n-glycols, except the peak activity occurred at 8 carbons, with significant activity extending to 14 carbons. PGE2 production was lower than arachidonic acid release, but followed the levels of released arachidonic acid with both alcohols and glycols. Cell viability measurements by trypan blue exclusion showed that these concentrations of the alcohols and glycols did not decrease the number of viable cells. An earlier report from our laboratory concerned the effect of long chain alcohols and glycols on hemolysis of erythrocytes in comparison with the membrane-active peptide Pyrularia thionin (Osorio e Castro et al., 1990). Again, a chain length dependence was observed, peaking at 12 carbons for both the alkanols and glycols. These and the above data on the effect of alcohols on synthetic phospholipid bilayers show that long chain alcohols which are effective in modifying the phospholipid bilayer structure with both cellular and synthetic membranes increase the action of soluble extracellular and endogenous PLA2 on the bilayers. The difference in the optimal chain length for the two systems involving PLA 2 activation is most likely due to the fact that added soluble PLA2 is acting on the outer leaflet while the endogenous enzyme is acting on the inner leaflet. There is further evidence for the activation of endogenous PLA2 by agents which affect the fluidity of cellular membranes. Hildebrandt and Albanesi (1991) showed a stimulation of membrane bound PLA2 in the secretory granules of the adrenal medulla by glycerol, ethylene glycol and poly(ethylene glycol). Other relevant examples of fluidity effects on endogenous membrane enzymes are the activation of phospholipase D in 3T3 fibroblasts by alcohols of varying chain length (2 to 9 carbons) (Kiss and Anderson, 1989) and activation of PLA 2 by changing the fluidity of liver plasma membrane (Petkova et al., 1987), as well as by the action of bile salts on rat jejunal brush-border membranes (Pind and Kuksis, 1988). The anticancer drug adriamycin stimulates PLA2 activity and this activity is related in part to its cytotoxicity. It modulates membrane structure and the fluidity of synthetic phospholipid bilayers (Murphree et al., 1981) and inserts into the hydrocarbon region of synthetic membranes containing CL and PC (Fiallo and Garnier-Suillerot, 1986; Constantinides et al., 1990). It also causes



clustering of CL (Huart et al., 1984). The drug is also active with soluble PLA2, stimulating the hydrolysis of pyrene-labeled phospholipid derivatives by porcine pacreatic PLA z (Mustonen and Kinnunen, 1991). It activates phospholipase C in sarcoma 180 cells (Posada et al., 1989) and also activates endogenous PLA2 in vivo (Ohuchi and Levine, 1978). DAG and inositol phosphates produced in the cell through the action of phospholipase C on phosphatidylinositol (PI) are very important second messengers in the cascade of events which stem from initial extracellular signals. Both of these products can give rise to or increase endogenous PLA 2 activity, the inositol phosphates by increasing intracellular Ca 2÷ concentrations and DAG by stimulating protein kinase C. A protein kinase C-independent mechanism for DAG activation of PLA2 activity involves a direct effect of DAG on cellular membranes. It is incorporated into phospholipid substrates, leading to membrane structural changes which facilitate interaction with the enzyme. Introduction of DAG into PE synthetic vesicles induces a lamellar-to-hexagonal transition at 5 mol% (30 mol% with PC) and disorders the phosphatidylserine (PS) lamellar phase (Das and Rand, 1984). These destabilizations of the bilayer are extensive enough to activate membrane enzymes. Dawson et al. (1983) report that addition of unsaturated 1,2- or 1,3-DAGs to synthetic bilayers of PC and PI enhances the activity of added PLA2, whereas hydrolysis of PE membranes is not affected. 3~p-NMR of the PC bilayers indicated that DAG causes the formation of a nonbilayer isotropic component which correlates generally with increased PLA 2 activity on the bilayer (Dawson et al., 1984). Further addition of DAG converts this structure into the H. phase with loss of enzyme activity. Calorimetric and enzymatic examination of monolayers and bilayers composed of DAG and PC showed the formation of either a complex or preferred packing array at 0.25 mol fraction of 1,3-dioleoylglycerol and at 0.8 mol fraction a nonlamellar phase was formed. Formation of the complex in mono- and bilayers followed the activity of porcine pancreatic PLA2 on the bilayer, suggesting a common effect of added DAG (Cunningham et al., 1989). Evidence that DAG stimulates endogenous PLA2 activity by a mechanism distinct from protein kinase C activation also comes from the experiments of Roldan and Mollinedo (1991). Rat spermatozoa, which lack protein kinase C show a stimulation of PLA2 activity when treated with DAG. Further evidence for cellular activation of endogenous PLAz by agents which perturb phospholipid bilayer structures is its activation in rat hepatocytes by carbon tetrachloride (Glende and Pushpendran, 1986) and in platelets by triethyl lead (Krug and Berndt, 1987) and tetraprenol (Nakano et al., 1989). A striking example of a similar phenomenon is the observation that the products of the hydrolysis reaction (fatty acid and lysophospholipid) promote activation of PLA 2 when present in the bilayer above a threshold concentration (Gheriani-Gruszka et al., 1988; Apitz-Castro et al., 1982; Bell and Biltonen, 1989). Indeed, this effect probably accounts for much of the very abrupt increase in PLA2 activity seen at the end of the lag phase during hydrolysis of large vesicles of saturated PC (Apitz-Castro et al., 1982; Bell and Biltonen, 1989). The possibility that activation resulting from the reaction products also reflects the formation of domains has been suggested (Jain et al., 1989; Bell and Biltonen, 1989). 4.1.2. M e m b r a n e Perturbation by Proteins and Peptides

The interaction of the phospholipids in cellular membranes with proteins and peptides is extensive and varied in nature. Hydrophobic regions of integral proteins are embedded in the bilayer and have varying amounts of polar regions of the proteins exposed and extending into the cytosol. The peripheral proteins, on the other hand, bind primarily by electrostatic forces to the membrane surface and extend to varying degrees into and perturb the bilayer. Perturbation of the membrane bilayer can also take place when extracellular proteins and hormones bind to protein receptors, changing their configuration and the nature of their association with surrounding phospholipids. Alternatively, amphipathic extracellular proteins and peptides can bind directly to the phospholipid headgroups of the bilayer with varying amounts of insertion into the bilayer. The possible consequences of peptide binding are those discussed above in Section 4.1. The bacterial antibiotic polymyxin is a positively charged (5 amino groups) decapeptide which activates extracellular PLA2 (Achari et al., 1987). Its binding to anionic dipalmitoylphosphatidic

Phospholipase A2 activity


acid (DPPA) phospholipid vesicles and the resulting changes in bilayer structure have been studied using diphenylhexatriene fluorescence polarization (Hartmann et al., 1978; Sixl and Galla, 1979, 1981). Polymixin induces one or two new transitions at lower temperature, indicative of the formation of fluid domains in the gel phase. In dipalmitoylphosphatidylcholine/DPPA vesicles lateral segregation of DPPA was observed, implying large domains of DPPA which bind the polymyxin (Galla and Trudell, 1980). The strongly basic synthetic polypeptide polylysine of MW 30,000 induces PLA2 activity in Swiss 3T3 fibroblasts (Shier et al., 1984). The activated enzyme is on the cell surface and is Ca 2+dependent. Other synthetic polycations gave the same response, indicating a generalized interaction with a negatively charged site on the membrane. Experiments performed with synthetic PC/PS mixtures show that polylysine induces a phase separation (Hartmann and Galla, 1978) and also has an effect on the tm of PS (Bonnet and Begard, 1984). With CL, added polylysine stabilizes the bilayer struture, which would explain its ability to facilitate fusion of such bilayers (Bloom and Smith, 1985). With PE membranes stabilized by 33 mol% CL, addition of polylysine induces the formation of the HH structure (de Kruijff and Cullis, 1980a). Myelin basic protein isolated from the myelin sheath induces a lateral phase separation in phosphatidylglycerol PG/PC synthetic bilayers with an increase in order parameter (Boggs and Moscarello, 1978). It also induces the formation of the HII structure in PS membranes (Smith et al., 1983) and decreases the quadrapole splitting in PG membranes, indicative of a disordering effect on the bilayer (Sixl et al., 1984). The data indicate that myelin basic protein penetrates further into the bilayer than does cytochrome c (Smith et al., 1983; Keniry and Smith, 1980). Since myelin basic protein can modulate the effect of PLA2 added to synthetic bilayers containing varying amounts of sulfatide and galactocerebroside (Bianco et al., 1992) and can change the intermolecular organization of such bilayers (Maggio and Yu, 1989), this difference in penetration may explain the difference in the abilities of the two strongly binding peptides to activate PLA2. The binding of a positively charged amphipathic peptide to, and partial insertion into, a cellular membrane bilayer will perturb the bilayer structure but may not necessarily activate an endogenous PLA2. The physical and biochemical consequence of cytochrome c interaction with synthetic and cellular membranes has been extensively studied (Devaux and Seigneuret, 1985). With synthetic bilayers cytochrome c causes a clustering of CL and exclusion of cholesterol from the lipid domain (Birrell and Hayes, 1976; Brown and Wuthrich, 1977). With mixtures of PC and PA, cytochrome c induces a segregation of the PA (Mustonen et al., 1987) and it induces the formation of the nonbilayer H . structure in CL/PE mixtures (de Kruijff and Cullis, 1980b). In spite of these extensive modifications of membrane phospholipid structure, cytochrome c does not activate an endogenous PLA2 in 3T3 cells as measured by release of arachidonic acid loaded into the cells (Evans, 1990). 4.2. P L A 2 ACTIVATION BY PLANT AND VENOM TOXINS

Whereas the PLA2-activating agents described above are mostly regulatory in nature, leading to the production of both arachidonic acid and prostaglandins in the cell, the group of peptides considered in this section activate extracellular and endogenous PLA 2 as a mechanism to inhibit or kill target cells. We will focus on three toxins: the bee venom component melittin, cardiotoxin (CTX) from cobra snake venoms and Pyrularia thionin (P. thionin), a plant toxin. Extensive experimentation has been done with both melittin and CTX in terms of their synergistic effect on PLA2 action with synthetic bilayers and cellular membranes as well as the physical consequences of their interactions with synthetic bilayers. Both melittin and CTX are components of venoms which also contain PLA2 and thus one of their functions in the venom is to combine with and disrupt the membrane structure to facilitate membrane breakdown by the venom or endogenous PLA2. There is always some degree of contamination of the purified venom toxins by venom PLA2 and studies involving the use of CTX and melittin must be carefully evaluated (Fletcher et al., 1991a). Since P. thionin preparations are not contaminated by PLA2 (Angerhofer et al., 1990; Osorio e Castro and Vernon, 1989), this toxin is well suited for such studies. An extensive series of experiments from the laboratory of Shier concerns the cytotoxic action of many toxins (Shier, 1980), relating these to the activation of different PLA2 enzymes in mouse 3T3 fibroblasts, the effect of C a 2 + on the activity of these enzymes and the separate effects of



increased cellular Ca 2+ on cell killing. PLA2 activation is involved in cell killing, since the two progress in parallel (Shier and DuBourdieu, 1985). As usual, these toxins are also hemolytic. The toxins phallolysin from the mushroom A m a n i t a phalloides (Shier and Trotter, 1980) and the toxin from Portugese Man-O-War stinging cells are both glycoproteins which likely activate endogenous PLA2 by some method other than membrane perturbation. Prymnesin, produced by the phytoflagellate P r y m n e s i u m p a r v u m is more soluble in organic solvents than in water and could act directly on cell membranes. Two of the toxins studied by Shier, melittin and cardiotoxin, are discussed in detail below. Another toxin, staphylococcal delta toxin, is an amphipathic polypeptide which exists as an aggregate of two subunits (Durkin and Shier, 1981) and resembles melittin in its mode of action. It also is hemolytic, activates endogenous PLA2 activity and is cytotoxic against a wide variety of cells. The CTX studied by Shier (designated as direct lytic factor) was from the African Ringhals cobra and of the toxins studied was the only one to activate a PLA2 which was not further activated by Ca 2+, a property of PLA2 from lysosomes. A hemolytic toxin (H-toxin) isolated from Clostridium septicum has been described (Takano et al., 1990). In addition to its hemolytic activity with rabbit erythrocytes, it also activates an endogenous PLA2. 4.2.1. Melittin Melittin is a 26 amino acid peptide which comprises almost half of the dry weight of bee venom. It has no negative charges and has positive charges at the N-terminus, Lys-7 and on four of the six C-terminus amino acids. Consequently melittin has a structure resembling a surfactant and it reacts readily with membranes. In aqueous media it forms a tetramer. It is generally toxic to cells, induces hemolysis of erythrocytes and enhances the activity on synthetic bilayers of isolated bee venom PLA2 (Yunes et al., 1977; Mollay and Kreil, 1974), but not pancreatic PLA2 (Conricode and Ochs, 1989). It also greatly enhances endogenous PLA 2 activity in many tissues, as described below. Melittin is cytotoxic to cells and has been extensively used as a biochemical tool to study cellular systems, giving varied responses depending on the concentration (Shier, 1983). At low concentrations it makes cell membranes permeable to Ca z+ ions, leading to cell death and over a narrow range of intermediate concentrations it appears to activate an endogenous PLA 2. At high concentrations it acts as a typical cationic detergent, causing rapid dissolution of cell membranes into mixed micelles. Melittin binding to phospholipid liposomes results in a blueshift of its fluorescence spectrum, indicating insertion of the Trp 19 into the bilayer and in the presence of salts it binds to phospholipids whose head groups differ in net charge (Dufourcq and Faucon, 1977). Thus, nonpolar interactions between the nonpolar portion of melittin and the hydrocarbon region of lipid bilayers are important. Using diphenylhexatriene fluorescence polarization, Bernard et al. (1982) showed that the phase transition of negatively charged phospholipids treated with melittin varied with both charge and chain length. Their data were interpreted in terms of formation of melittin-phospholipid domains, with both electrostatic and hydrophobic interactions being important. The binding of melittin to mixed PG/PC membranes was studied by circular dichroism and 2H-NMR (Beschiaschvili and Seelig, 1990), indicating that about 50% of the melittin surface was embedded in a hydrophobic environment. This interaction of melittin with membranes stabilizes the bilayer structure, as shown by an increase in order parameter with DMPC vesicles using diphenylhexatriene emission anisotropy (Bradrick et al., 1987) and an inhibition of the nonbilayer HH structure in PE phospholipid model systems (Batenberg et al., 1988). The interaction of melittin with the hydrocarbon region of phospholipid bilayers to form clusters (Posch et al., 1983) or domains (Bernard et al., 1982) leads to major changes in membrane morphology, including the vesicularization of multibilayers, fusion of small lipid vesicles and fragmentation into discs and micelles (Dufourcq et al., 1986). Taken together, the data suggest that melittin's facilitation of PLA 2 activity stems from its ability to dissolve into the hydrocarbon region of the bilayer, the subsequent formation of melittin-phospholipid domains and consequent alteration of the membrane surface. There are numerous reports of an activation by melittin of endogenous PLA2 activity in various cells and tissues. Fletcher et al. (1990) give references for reported stimulation of an endogenous PLA2 as the cause for physiological responses in rat anterior pituitary cell cultures, human

Phospholipase A2 activity


erythrocytes, anterior pituitary tissue, pancreas, rat adenohypophysis, vascular endothelial cells and human leukocytes and platelets. Other reported stimulations include mouse 3T3 fibroblast cells (Shier, 1979), placental tissue (Zeitler et al., 1991), rat pituitary cells (Kiesel et al., 1987) and rat parotid gland (Mizuno et al., 1991). The interpretation that these responses are due to activation of an endogenous PLA2 by melittin may or may not be correct, however, since melittin preparations contain varying levels of contaminating PLA2 (Shier, 1983). It is impossible using current techniques to prepare melittin samples completely free of contaminating PLA2 and optimal conditions reduce the contamination to 0.1% or less (Dufourcq et al., 1986). This was pointed out by Metz (1986), who showed that commercial melittin samples are contaminated by PLA2 and bee venom PLA2 itself could cause the release of insulin from rat islets, a response attributed to activation of endogenous PLA 2. This is treated in more detail by Fletcher et al. (1990), who report that PLA2 contamination can account for 75% of the activity of melittin on epithelial cells. They suggest treating melittin samples with p-bromophenacyl bromide (p-BPB) to inactivate contaminating PLAy Alternatively, synthetic melittin could be used. In a later study, Fletcher et al. (1991 a) examined the effects of contaminating PLA2 on the action of CTX and melittin on skeletal muscle phospholipids. To show the effect of contaminating PLA 2 the toxin fractions were treated with p-BPB to inactivate PLA 2 and the activity of treated melittin fractions was compared to that of synthetic melittin. Whereas data obtained with untreated toxin fractions would indicate activation of an endogenous PLA2, the treated melittin and synthetic melittin behaved similarly, activating primarily a phospholipase C. These data show that p-BPB treatment is sufficient to inactivate contaminating PLA 2 in the systems studied. Although it is clear that melittin facilitates interaction of PLA2 with phospholipid bilayers, it appears that in most cases the melittin-associated PLA 2 activity reported with cellular membranes comes from a contaminating PLA2 rather than an activation of an endogenous PLA 2. 4.2.2. Cardiotoxin Cardiotoxin (CTX) is a small basic membrane-active peptide which like melittin has been reported to activate endogenous PLA 2 activity. For reviews on CTX properties and physiological effects, see Harvey (1985, 1991). Shier (1979) used CTX (designated direct lyric factor) from African Ringhals cobra venom to stimulate PLA 2 activity with mouse 3T3 fibroblasts and Narita and Lee (1970) reported a similar activity with a CTX from Naja naja atra. CTX is the name accepted for this family of peptides because of its direct effect on heart muscle, causing depolarization and prolonged muscular contractures. Investigation of the CTXs has focussed on the two primary activities of the peptide, depolarization of muscle cells and hemolysis of erythrocytes. As with melittin, the hemolytic activity of CTX is dramatically increased in the presence of PLA2, a synergism which greatly increases the cytotoxicity of the cobra venoms, but this makes it very difficult to separate CTX effects from those of the contaminating venom PLA 2. Most (if not all) CTX preparations are contaminated by some level of PLA 2 and reported activations of endogenous PLA 2 by CTX must be considered in light of this contamination. A dramatic increase in hemolysis rates induced by several CTXs is produced by addition of venom PLA 2 to CTX (Lankisch et al., 1971; Louw and Visser, 1978; Harvey et al., 1983). Although most CTXs no longer produce hemolysis when the contaminating PLA2 is removed, the toxins from Naja naja kaouthia (Jiang et al., 1989; Fletcher et al., 1991a) and Naja naja siamesis (Hodges et al., 1987) do retain a direct hemolytic activity. A synergism is also shown in vivo, since injecting mice i.v. with PLA 2 enhances the lethality of injected CTX (Bougis et al., 1987). A synergism is not as readily shown for the effects of CTX on muscle cells, but high levels of PLA 2 do enhance the ability of CTX to block action potentials (Chang et al., 1972) and muscle contractures (Fletcher and Lizzo, 1987). Because of the PLA 2 contamination of CTXs prepared from cobra venoms, experiments reporting an activation of an endogenous PLA2 by CTX are not definitive, as suggested by Fletcher et al. (1991b) and Harvey (1991). A claim for PLA2 activation would require proof of noncontamination or inactivation of the contaminating enzyme by a treatment such as with p-BPB, as performed by Fletcher et al. (1991b). Using this procedure to inhibit contaminating PLA2 in CTX from Naja naja kaouthia, it was shown that CTX activates a phospholipase C and not PLA2 in



muscle cells. Similar data were obtained for the CTX from Naja naja atra, which has a higher level of PLA2 contamination (Fletcher et al., 1991a). Neither toxin was active in the release of fatty acids from aged erythrocytes if the preparations were first treated with p-BPB. CTX from Naja naja atra, reported to be free of contaminating PLA2 by pH-stat titration and the indirect hemolytic method, potentiates the aggregation of platelets as well as activation of platelet PLA2 (Teng et al., 1984). The methods used, however, are not sensitive enough to detect levels of contaminating PLA2 that would be active in conjunction with CTX. However, treatment of the platelets with p-BPB blocked the potentiating effect of CTX, indicating the effect was due to activation of an endogenous PLA 2. There has not been as much work done on the synergism between PLA 2 and CTX as between the enzyme and melittin with synthetic membranes, but such a synergism has been shown. Gasanov et al. (1991) reported a synergism between cobra venom PLA2 and a CTX (called a cytotoxin) isolated from Middle-Asian cobra Naja naja oxiana. A later study involved CTX from Naja naja kaouthia and two PLA2 species from Crotalus molossus molossus.* Addition of CTX increased the activity of both PLA 2 enzymes on liposomes of PC with 25 and 50% of the acidic phospholipids PS, PA and CL, but not with PE. Other experiments with PC membranes containing 25 mol% PS or PA have shown that small amounts of the Asian cobra CTX induce the formation of phospholipid clusters having a high level of molecular order as well as clusters with a high level of phospholipid fluidity (Gasanov et al., 1990a,b) and increasing membrane fluidity facilitates the enhancing effect of CTX on PLA 2 activity (Gasanov and Aripov, 1991). A similar effect was observed for CTX enhancement of PLAz activity on liposomes containing 50 mol% CL, which could be correlated with the ability of CL to induce nonbilayer structures in natural membranes (Batenberg et al., 1985). There is an extensive literature on the physical consequence of CTX interaction with phospholipid bilayers. CTX II from Naja rnossambica mossambica binds readily to phospholipid vesicles containing PS, PA or PI, resulting in changes in tryptophan fluorescence, indicating insertion of a part of the CTX molecule into the hydrocarbon region of the membrane. It also causes a phase separation in PC/PS and PC/PA vesicles (Dufourcq and Faucon, 1978; Vincent et al., 1978). By diphenylhexatriene fluorescence polarization measurements, the same CTX was shown to increase probe motion in the gel phase and decrease it in the fluid phase of membranes containing acidic phospholipids (Faucon et al., 1981, 1983). With DMPC/PA membranes, the zwitterionic phosholipids were squeezed out of the PA-peptide domains (Faucon et al., 1983). The primary result of membrane-CTX interaction is most likely the insertion of a portion of the first loop of the peptide into the bilayer as shown by fluorescence changes of the Trp located in this loop (Bougis et al., 1983; Dufourcq et al., 1982; Gatineau et al., 1987). Using spin label probes, the CTX isolated from Asian cobra venom (designated as cytotoxin Vc5) was shown to insert partially into phospholipid bilayers to cause an increase in the probe order parameter for PA liposomes (Aripov et al., 1984). This CTX also interacts with PC/PS and PC/PI liposomes to cause aggregation, increased permeability and enhanced fusion (Aripov et al., 1989). Other effects of CTX with PC/PA liposomes were a phase separation and the formation of a new lipid phase observed by differential scanning calorimetry (Aripov et al., 1987). Insertion of CTX into a PC bitayer with 10% PA was shown by X-ray small angle scattering (Oimatov et al., 1986). All of the data cited above for CTX emphasize the avid binding of CTX to phospholipid bilayers containing acidic phospholipids, the insertion of the first loop into the bilayer to cause profound changes in the phospholipid structure, including phase separation, clustering of phospholipids (domain formation), lateral segregation of phospholipids and the ability to induce membrane fusion. The latter ability is usually associated with nonbilayer structures such as the H , structure. These modifications by CTX to the bilayer structure of synthetic membranes facilitate PLA2 activity. Similar bilayer changes induced by CTX in cellular membranes could cause an activation of endogenous PLA2 enzymes. This is most likely the case, but such has not been definitively shown to date because of the universal contamination of isolated and purified CTX with varying amounts of PLA2 from the cobra venom. A systematic study needs to be done with CTX having no *Gasanov, S. E., Rael, E. D. and Vernon, L. P. Enzymatic activity of two phospholipases A 2 from Crotalus molossus molossus venom on unilamellar liposomes modified by membrane- active polypeptides. Toxicon, submitted.

Phospholipase A 2



demonstrable PLA 2 contamination by the most sensitive techniques, with CTX preparations in which the synergistic activity of contaminating PLA2 is inhibited by treatment with agents such as p-BPB, or preferably with synthetic or cloned and expressed CTX. One reason for believing that CTX induces endogenous PLA2 activity is the fact that P. thionin, discussed below, does activate endogenous PLA2 activity and has no contaminating PLA z. In all comparisons made to date, P. thionin behaves similarly to Naja naja kaouthia CTX in terms of their binding properties to erythrocytes and the resulting hemolysis as well as the inhibition of binding by Ca 2+ at 10 mM. Furthermore, the two strongly basic peptides bind competitively to the same site on the erythrocyte membrane. However the data of Fletcher et al. (1991a,b) show that with muscle cells, CTX activates a phospholipase C that is significantly more active than any endogenous PLA2. This emphasizes that although release of loaded arachidonic acid is usually taken as an indication of PLA 2 activity, the combined action of phospholipase C and diacylglycerol lipase can also cause the release of arachidonic acid. Definite proof of PLA2 activation requires the determination of both fatty acid release as well as lysophospholipid formation in the bilayer. 4.2.3. Pyrularia Thionin A small, strongly basic peptide with cytotoxic and hemolytic activity was isolated from nuts of Pyrularia pubera. It contains 47 amino acids with four disulfide bonds and the location of these bonds and the amino acid sequence place it in the family of plant toxins known as thionins (Vernon et al., 1985). A similar toxin from mistletoe, viscotoxin, has amino acid sequence homology, but has only 3 disulfide bonds. The toxins as a family are very stable, low MW, water soluble and cytotoxic to bacteria, fungi, yeast and mammalian cells. Some are hemolytic and in general, they affect membranes to make them leaky and permeable to small ions. A review of the thionins has recently appeared (Bohlmann and Apel, 1991). Since more has been done with P. thionin in the area of PLA2 activation and membrane interactions, this review will concentrate on this thionin. A very important feature of the P. thionin preparations used to date is the lack of any PLAz contamination. This was shown by the lack of pH change using egg yolk PC as substrate and lack of uptake of oxygen in the lipoxidase assay (Osorio e Castro et al., 1989). The thionin has also been studied by Shier and his group, who reported no detectable phospholipase activity on radiolabeled PC in an assay that would have detected 20 pmol of PC hydrolyzed per min (Angerhofer et al., 1990). This lack of contaminating PLA 2 makes P. thionin ideally suited for studying activation of endogenous PLA2. A second very favorable feature of the peptide is that in terms of interaction with erythrocytes to produce hemolysis, P. thionin and CTX from Naja naja kaouthia behave almost identically. The two peptides bind to erythrocytes in a competitive fashion with a Km in the hemolysis reaction of 1.6/tM for P. thionin (Osorio e Castro et al., 1989) and 0.7 /tM for CTX (Osorio e Castro and Vernon, 1989). The hemolytic potential of P. thionin is enhanced synergistically by added PLA2 (Osorio e Castro et al., 1989) and melittin (Osorio e Castro and Vernon, 1989) in a manner reported above for CTX. Further comparison of the two peptides show that for the hemolytic activity induced by both peptides, removal of C a 2 + with EGTA stimulates the reaction and addition of Ca 2+ at high concentrations (10 mM) inhibits the reactions (Vernon and Rogers, 1992a). A companion study on binding of both peptides to erythrocytes showed a similar effect of EGTA treatment and added Ca z+ on the binding properties (Vernon and Rogers, 1992b). The number of binding sites ranged from 0.7 to 1.7 x 105 for P. thionin and 0.82 to 1.6 × 105 for CTX. The binding constants are in the same range as the Km values and are in the range observed for binding of CTX to synthetic membranes. P. thionin and cobra venom CTXs have been directly compared for their ability to depolarize cultured chick myotubes. Both were active, but with different potencies. Their depolarizing effects were not due to activation of endogenous PLA2 (A. L. Harvey, personal communication) and probably reflect a direct effect on the membrane. Attempts to find a protein receptor for P. thionin have been unsuccessful (Evans, 1990) and in light of the facile interaction of P. thionin and CTX with phospholipid bilayers leading to membrane perturbation, we believe both peptides bind to specific phospholipid domains on cellular membranes, cause alterations in phospholipid structures which facilitate the observed cellular responses for both peptides. These data, coupled with specific effects of P. thionin on liposomes



containing PS, led us to suggest that the binding site for both P. thionin and CTX on cellular membranes involves primarily the acidic phospholipid PS in some specific domain. P. thionin does induce a PLA2 in NIH3T3 fibroblast cells, as indicated by release of radiolabeled arachidonic acid from loaded cells (Angerhofer et al., 1990). A release of radiolabeled palmitic acid was also stimulated by P. thionin, indicative of an activation of a phospholipase At. Arachidonic acid release was inhibited by added p-BPB and this coupled to the lack of contaminating PLA 2 activity in the P. thionin preparation shows activation of an endogenous enzyme. The salient features of the cellular responses to P. thionin included a time lag of about 20 min for both arachidonic acid release and the parallel cell killing, a lag in the dose response up to 10 #g/mL for both activities, a lack of Ca 2+ sensitivity at low concentrations indicative of a lysosomal enzyme and a similar activation by purothionin, but not by phoratoxin or crambin. Similar experiments have been performed in our laboratory (L.V.) with similar results, except that we observed a Ca 2+ sensitivity for the enzyme, being stimulated by 1.8 mM Ca 2+ (Evans, 1990). Activation of a PLA2 was shown by an inhibition of arachidonic acid release with dexamethasone but not the phospholipase C inhibitor neomycin. There was no involvement of G proteins since neither pertussis nor cholera toxin had any effect. However, whereas arachidonate release from 3T3 cells shows a lag of about 20 min at 100/~g/mL (about 20 #M) of P. thionin, there were several more rapid responses observed in other cell types. These included a membrane depolarization in mouse P388 cells and frog sartorius muscle (Evans et al., 1989), an influx of Ca :+ into P388 cells, the release of 5~Cr from loaded P388 cells and the formation of membrane btebs with P388 cells. Another rapid response reported earlier is a decrease in membrane fluidity of erythrocyte ghosts by P. thionin (Osorio e Castro et al., 1990). The activation of endogenous PLA2 is a general response, observed by us with Rat2 cells, mouse P388 and H160 cells, chicken embryo fibroblasts and erythrocytes (Evans, 1990). Another study involved the activation of phospholipase activity and liberation of prolactin and growth hormone from rat anterior pituitary cells by P. thionin (Judd et al., 1992). The thionin increases the release of arachidonic acid from loaded cells as well as increasing cellular levels of lysophospholipids, suggesting activation of PLA 2. It also increases the release of stearate, indicating an activation of phospholipase Aj, as was the case with 3T3 cells (Angerhofer et al., 1990). Interestingly, the Ca 2+ channel blocker methoxyverapamil and also dopamine decreased the amounts of prolactin and growth hormone released, without affecting the arachidonic acid release, indicating that a Ca 2+ influx was not the stimulus for PLA2 activation. This coupled with the lack of action by pertussis and cholera toxins in 3T3 cells lends more support for a membrane perturbation as the primary cause of PLA 2 activation by P. thionin. P. thionin reacts readily with phospholipid bilayers containing acidic phospholipids. The structural aspects of the interaction have been studied by measuring phosphorescence quenching, ESR spectroscopy with spin labels and tH- and 3tp-NMR at different phospholipid compositions.* With PC bilayers containing CL or PI, P. thionin induces an increase in membrane viscosity and at higher P. thionin concentrations nonbilayer structures are formed. PC bilayers containing PS respond differently in that the nonbilayer structures form at lower P. thionin concentrations and the membrane viscosity decreases. P. thionin also induces membrane fusion, indicative of formation of an H n structure in the membrane. This unique response to synthetic membranes with PS argues for a role for PS in cellular membrane responses to P. thionin (and also for CTX). P. thionin action on cell membranes can be considered in the light of four responses which are temporally separated: (1) P. thionin binding, (2) perturbation of the phospholipid structure of the bilayer, (3) depolarization of the membrane and an associated opening of a C a 2+ channel and (4) activation of an endogenous PLA2 which leads to membrane disruption and cell death. The binding process, which has been studied extensively with erythrocytes (Vernon and Rogers, 1992b) involves binding to a phospholipid site on the membrane, most likely containing PS. Both P. thionin and CTX bind competitively to the same site with similar binding properties. Ca 2+ at high concentrations (10 mM) inhibits the binding. *Gasanov, S. E., Vernon, L. P. and Aripov, T. F. Modification of phospholipid membrane structure by the plant toxic peptide pyrularia thionin. Biochim. biophys. Acta, submitted.

Phospholipase A2 activity


The second phase involves perturbation of the phospholipid bilayer. This is shown directly by the earlier report of a P. thionin-induced decrease in membrane fluidity of erythrocyte ghosts (Osorio e Castro et al., 1990) and also by depolarization of the cellular membrane (Evans et al., 1989), by rapid changes in membrane permeability as shown by chromium release experiments and by the formation of membrane blebs, all in P388 cells (Evans, 1990). All of these responses are rapid, proceeding without the time lag observed for PLA2 activation (Angerhofer et al., 1990). The third phase involves opening of a Ca 2÷ channel and an influx of Ca 2+. This has been shown with mouse P388 cells (Evans et al., 1989) and with rat anterior pituitary cells in which the channel blocker methoxyverapamil and dopamine, which closes Ca 2÷ channels (Login et al., 1988), inhibit the P. thionin-induced release of growth hormone from the cell (Judd et al., 1992). The speed of all three initial responses to P. thionin sets them apart from the fourth phase, PLA2 activation, which shows a distinct 20 min time lag (Angerhofer et al., 1990). The nonlinear dose response for P. thionin-induced PLA2 activity shows that the peptide is only marginally effective below 10/~g/mL (about 2/~M). The dose-response and the time lag can be explained in terms of the known kinetic properties of PLA 2 interaction with synthetic phospholipid bilayers discussed above. With such bilayers a lag period is observed which represents a slow accumulation of the hydrolysis products lysolecithin and fatty acids. Once these build up sufficiently to affect the membrane structure in a major way, the fast phase of the enzyme reaction begins. Hypothetically, P. thionin would initially perturb the bilayer in cells to cause a minor and barely perceptible activation of endogenous PLA2. When the products of this initial reaction accumulate and perturb the membrane further, the major activity of the endogenous enzyme is expressed. The fast phase of PLA2 activation would be time- and dose-dependent, which is observed.


4.3.1. General Hypothesis The extreme complexity of the various phenomena described above has impeded the understanding of the molecular details of how the state of activity of PLA 2 is altered by the membrane environment. Most investigations on the mechanism of PLA 2 activation have focused on the more simple artificial systems. Several hypotheses for the effects of the bilayer on PLA2 activity have been proposed. Terms such as 'quality of the interface' have been introduced to explain the observations that PLA2 prefers a surface that is not homogeneous in structure and conformation (Verger et al., 1973). The assumption has generally been that defects or changes in the quality of the interface somehow put the bilayer in a configuration that makes it susceptible to hydrolysis or is conducive to the active state of the enzyme. A novel hypothesis has been proposed in which it is not the state of the bilayer per se that promotes activation of PLA 2, but rather it may be the dynamics of fluctuations of bilayer structures that promotes activation (Gheriani-Gruszka et al., 1988; Lichtenberg et al., 1986; Bell and Biltonen, 1992). A general hypothesis for the specific effect of a threshold concentration of reaction products to promote a sudden increase in PLA 2 activity has been formulated into a mathematical model (Bell and Biltonen, 1992). In the model, the reaction products cause a hypothetical cooperative transition in bilayer structure that is coupled to activation of the enzyme. Experimental evidence for such a transition has been obtained using fluorescent membrane probes (Bell and Biltonen, 1989; Jain et al., 1989). However, a detailed molecular description of the physical nature of this putative transition and the extent to which it relates to the other stimulatory membrane perturbations has not yet been elucidated. Various models describing hypotheses for the molecular nature of the actual activation process have also been described. Some of these models attempt to explain the nature of the dependence of activity on the state of aggregation of the lipid substrate, some address the apparent time-dependent activation and some attempt to explain both. Whether both types of observations represent the same activation phenomenon is not known, but it is attractive to hope that some general model that accounts explicitly for all activation phenomena could eventually be found. The current models can be classified as the following three types: binding models, monomer models and dimer models (Bell and Biltonen, 1992). JPT 5 4 / ~ E



4.3.2. Binding M o d e l In general, the binding model assumes that PLA2 activity is limited by the ability of the enzyme to interact with and bind to the surface of the membrane (Jain et al., 1982, 1988, 1989; Bell and Biltonen, 1992). The evidence that has been introduced to support this model consists mostly of correlations between the level of initial PLA2 activity and the apparent binding affinity of PLA2 for various bilayers. Both the binding and activity of the enzyme are reported to be high with anionic phospholipid bilayers and both are reported to be low with zwitterionic bilayers (Jain et al., 1982, 1986, 1989). Also, increases in binding have been suggested to explain the sudden increase in activity at the end of the lag phase when the mole fraction of reaction product has reached a threshold value (Jain and Berg, 1989; Apitz-Castro et al., 1982; Jain et al., 1989). It is proposed that the presence of a critical level of negative charge in the bilayer is necessary for proper PLA2 binding (Jain and Berg, 1989; Jain et al., 1989). This negative charge would be provided either by the presence of anionic phospholipid or by the fatty acid that accumulates in the bilayer during the lag phase of hydrolysis of zwitterionic phospholipid. While the above mentioned data do support the binding model, they do not exclude other models. For example, if certain configurations of the bilayer promote activation of the enzyme, it is required thermodynamically that they also enhance the binding affinity of the enzyme tbr those configurations of the bilayer. Also, while some kinetic results support the binding model, others seem to exclude it (Bell and Biltonen, 1992). Specifically, the binding model predicts that the apparent time-dependent activation at the end of the lag phase during hydrolysis of zwitterionic phospholipid vesicles would become increasingly less abrupt as the vesicle concentration is increased (Bell and Biltonen, 1992). Experimentally, that prediction is not corroborated (Bell and Biltonen, 1992). Also, the concept of the requirement for negative charge in the bilayer is probably over-simplistic, since instantaneous activity (i.e. lag time equals zero) can occur without introduction of negative charge into the bilayer. Examples of such are the observations that PLA 2 is active without a lag phase toward small unilamellar vesicles of PC (zwitterionic) in the gel phase (Menashe et al., 1986) and that lysolecithin added directly to the outer monolayer of zwitterionic vesicles apparently reduces the lag phase to zero (Bell and Biltonen, 1992; Jain and De Haas, 1983).

4.3.3. M o n o m e r M o d e l s These are models in which activation represents an additional change in the interaction between the enzyme and the substrate after binding of the PLA 2 to the membrane surface and the active form of the enzyme is a monomer. Some such models suggest that the activation step involves a change in enzyme conformation (Verger et al., 1973; Tinker and Wei, 1979; Bianco et al., 1991). Others propose that the enzyme does not change conformation, but that it is the conformation of the substrate molecules within the bilayer that must change for activation to occur (Barlow et al., 1988; Thuren, 1988; Achari et al., 1987). These two possibilities are not mutually exclusive and are very difficult to distinguish experimentally, since they behave identically in kinetic experiments (Bell and Biltonen, 1992). One specific model states that the enzyme binds to the membrane surface and must then penetrate into the bilayer in order to catalyze hydrolysis of the phospholipid (Verger et al., 1973). The penetration step is proposed to be slow under conditions when time dependence is observed. Conditions that promote rapid activation would presumably favor the penetration of the enzyme. A second monomer model proposes that PLA2 assumes an active conformation upon binding to the membrane surface; the enzyme exchanges rapidly among vesicles under conditions of rapid hydrolysis and exchanges slowly when hydrolysis is slow (Tinker and Wei, 1979). The products of the reaction are predicted to promote high activity by increasing the rate of desorption of the enzyme from the vesicles. This model predicts that the lag phases that occur during hydrolysis of zwitterionic vesicles will be preceded by a transient burst of rapid activity (Tinker and Wei, 1979). Such a burst is sometimes seen, but does not appear to be a general property of the lag phase of hydrolysis time courses (Romero et al., 1987). Also, the data used to support the binding model support the conclusion that the enzyme binds better in the presence of the reaction products rather

Phospholipase A 2 activity


than worse, as is suggested by this model (Jain and Berg, 1989; Apitz-Castro et al., 1982; Jain et al., 1989). Evidence from X-ray crystallography data using structural analogs of the hydrolysis transition state of the phospholipid suggest that a major conformation change of the enzyme backbone is not required for the enzyme to be active (White et al., 1990). The investigators proposed instead that activation represents conditions at which the substrate monomers within the bilayer would be able to insert into the active site on the enzyme. Therefore, instead of the enzyme penetrating into the bilayer, the lipids are proposed to penetrate into the active site of the enzyme. 4.3.4. D i m e r M o d e l s Some of the PLA 2 from snake venoms are dimers in the purified state (Verheij et al., 1981). The possibility that the active form of PLA 2 is an enzyme dimer was proposed early for the enzyme from the venom of Crotalus adamanteus (Wells, 1971). This hypothesis was supported by kinetic studies on PC monolayers (Shen et al., 1974). A dimer model in which the binding of phospholipid to one of the enzyme monomers serves to activate catalysis by the sister monomer was subsequently proposed based on data for the Naja naja naja enzyme (Roberts et al., 1977). This 'dual-phospholipid' model has been supported by a variety of kinetic experiments with mixed micelles and other biochemical techniques such as chromatography, centrifugation and cross-linking (e.g. Lombardo and Dennis, 1985; Hazlett and Dennis, 1988). Another analogous model has recently proposed that two phospholipid molecules are required for the enzyme to be catalytically active (Yokoyama and Kezdy, 1991). The support for this model was kinetic and again the assumption was made that the enzyme is active as a dimer. As mentioned above, it has been reported that PLAz can become acylated during the hydrolysis of phospholipid substrate (Cho et al., 1988; Tomasselli et al., 1989). The acylation appears to result from a reaction between the fatty acid produced during the hydrolysis and specific lysine residues in the enzyme. This acylated enzyme is stable as a dimer and appears more active than before acylation. Furthermore, under certain conditions the activation of the virgin enzyme was time-dependent, but after acylation, the enzyme was active without a lag (Cho et al., 1988). Recent reports, however, suggest that acylation is not necessary for high catalytic activity to be expressed (Noel et al., 1991). Finally, kinetic studies of the apparent time-dependent activation of PLA2 on the surface of phosphatidylcholine vesicles as a function of enzyme and vesicle concentration also suggest that activation of the enzyme indeed involves dimerization (Romero et al., 1987; Bell and Biltonen, 1992). Specifically, it was found that the length of the lag phase varies with vesicle concentration such that it decreases with increasing vesicle concentration, reaches a minimum value and increases linearly with further elevation of vesicle concentration. Theoretical arguments showed that such a result is not possible under conditions at which the various states of the enzyme are always at equilibrium during the hydrolysis reaction (Bell and Biltonen, 1992). The most direct explanation for these kinetic data was a model in which active dimer PLA2 accumulates slowly during the lag phase and rapidly in the presence of reaction products. The dimer models have been criticized by reports suggesting that the enzyme may be fully active as a monomer (Bukowski and Teller, 1986; Jain et al., 1991). One source of such criticism comes from centrifugation and fluorescence studies in which it was concluded that a dimer enzyme from Crotalus atrox dissociates into monomers upon interaction with phospholipid (Bukowski and Teller, 1986). However, as the authors stated, their experiments do not conclusively demonstrate whether it is the monomer or dimer form of the enzyme that is active. Interestingly, the X-ray crystallographic data for the dimer C. atrox enzyme suggests that the active site of the enzyme is occluded in the dimer structure (Achari et al., 1987). Therefore, if the dimer form is the active form, a rearrangement of the quaternary and or tertiary conformation would apparently be required for catalysis to occur. Indeed, we have found in our lab that lag phases do exist for the C. atrox enzyme prior to activation during hydrolysis of PC vesicles, suggesting that some sort of activation step other than or beyond dimerization is required. Such is also proposed in the dimer model described above (Bell and Biitonen, 1992; Shen et al., 1974).



Another criticism of the dimer hypothesis comes from kinetic studies with anionic phospholipid vesicles (Jain et al., 1991). These experiments used very low concentrations of enzyme relative to the phospholipid concentration. The conditions were chosen so that the probability was high that less than one enzyme molecule was present on each vesicle and the assumption was made that there was no exchange of enzyme among vesicles during the experiment. The enzyme appeared active in the time courses shown in that the rate of hydrolysis was greatest at initial time. Under such conditions, the total amount of lipid hydrolyzed was a linear function of enzyme concentration such that the amount hydrolyzed per enzyme was constant and appeared equal to the amount of lipid in the outer monolayer. It would appear from such studies that the enzyme was active at initial time as a m o n o m e r and that no activation occurred during the time course. However, this type of analysis has been criticized based on the assumptions required for the interpretation (Romero et aL, 1987). Nevertheless, even if these results argue that the m o n o m e r enzyme is active, they do not refute the possibility that the enzyme could form a more active dimer slowly (Romero et al., 1987; Cho et al., 1988; Tomasselli et al., 1989; Bell and Biltonen, 1992).

5. S U M M A R Y We have reviewed the extensive evidence that phospholipase A 2 is regulated by a variety of mechanisms in living cells. These mechanisms include the c o m m o n features of hormone signal transduction such as G protein involvement, Ca 2÷ fluxes and covalent modification. In addition, we have presented strong evidence to support the hypothesis that phospholipase A 2 activity is also modulated by alterations in the structure and dynamics of the phospholipid bilayer that serves as substrate for the enzyme. The enzyme is extremely sensitive to perturbations of the bilayer as well as being selective for which perturbation will activate the enzyme. Some of the exciting challenges of the future will be to understand the molecular details relating changes in bilayer structure to changes in enzyme activity and to apply this knowledge to the physiology and pathology of biomembranes. In a larger sense, the principles reviewed here apply also to other enzymes whose function depends on their association and interaction with cellular phospholipid bilayers.

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Phospholipase A2 activity


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Phospholipase A 2 activity


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Membrane structure, toxins and phospholipase A2 activity.

The phospholipid-hydrolyzing enzyme phospholipase A2 (PLA2) (EC exists in several forms which can be located in the cytosol or on cellular me...
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