Adamich, M

Dennis, EA

Adamich, Marina

Dennis, Edward A

eScholarship - University of California

UC San DiegoUC San Diego Previously Published WorksTitlePhospholipase A2 Action on Membrane Surfaces Effect of Ionic Strength, Triton X-100, and MelittinPermalinkhttps://escholarship.org/uc/item/9436b7n8JournalBiophysical Journal, 37(1)ISSN0006-3495AuthorsAdamich, MarinaDennis, Edward APublication Date1982DOI10.1016/s0006-3495(82)84653-5 Peer reviewedeScholarship.org Powered by the California Digital LibraryUniversity of CaliforniaPHOSPHOLIPASE A2 ACTION ON MEMBRANE SURFACESEFFECT OF IONIC STRENGTH, TRITON X- 100, AND MELITTINMARINA ADAMICHDepartment ofChemistry, Atlanta University, Atlanta, Georgia 30314 U.S.A.EDWARD A. DENNISDepartment ofChemistry, University of California at San Diego, La Jolla, California 92093 U.S.A.Phospholipase A2 has been used extensively as a probe forthe distribution of phospholipids in membrane surfaces.The implicit assumption has been that the activity andspecificity of the enzyme are dependent on the distributionof the phospholipids across the membrane. However, toachieve significant hydrolysis of intact erythrocytes,nonphysiological conditions and/or high concentrations ofenzyme are necessary (1). In previous studies using lowconcentrations of a highly purified homogeneous prepara-tion of phospholipase A2 from cobra venom (Naja najanaja) and metabolically competent human erythrocytes,we found that under initial rate conditions the enzyme isbarely active toward the outer surface of intact erythro-cytes, but that the ghosting procedure results in an enor-mous activation of the enzyme toward phosphatidyletha-nolamine (PE) and phosphatidylcholine (PC) (1, 2). Withphospholipid mixtures in Triton X-100 mixed micelles, wehave found that phospholipase A2 activation toward PE isdependent on the direct binding of specific phosphoryl-choline-containing activator lipids to the enzyme (3, 4).For a valid interpretation of the distribution ofmembrane phospholipids using phospholipase A2, keepingin mind possible phospholipid activation of the enzyme, wemust assess the cause(s) of the low activity of phospholi-pase A2 toward intact erythrocytes, and whether activationtoward the erythrocyte results from prehemolytic subtlealterations in the outer surface of the membrane or fromposthemolytic exposure of the lipids at the innermembrane surface. We have now examined the action ofcobra venom phospholipase A2 toward minimallyperturbed human erythrocytes to see if hydrolysis can beactivated in the absence of hemolysis.MATERIALS AND METHODSFresh, defibrinated human erythrocytes were prepared in varying ionicstrength solutions of NaCl in buffer (20 mM Hepes, pH 7.4 and 5 mMD-glucose) (1). Duplicate incubation mixtures were prepared in a totalvolume of 2.0 ml and included 0.5 ml packed erythrocytes (1 timolphospholipid), the same buffer, 10 mM CaC12, and an ionic strength of306 mosmol (measured) unless indicated otherwise. Samples were incu-bated for 1 min at 370C in a shaking water bath prior to the addition of 2lg enzyme purified as in previous studies (1). The reaction was quenchedafter 10 min with the addition of 0.3 ml 0.5 M EDTA and 0.5 mlmethanol. Following organic extraction, total and individual phospholipidconcentrations were determined by two-dimensional thin-layer chroma-tography and phosphorus analysis described previously (1). Where indi-cated, Triton X-100 (Rohm and Haas Co., Philadelphia, PA) or melittin(Sigma Chemical Co., St. Louis, MO) first treated with p-bromophena-cyl bromide (BPB) to inactivate any residual bee venom phospholipaseA2 (4) were included in the incubations. Hemolysis was determined onduplicate samples (not treated with EDTA and methanol) by the methodof Anderson et al. (5). For the measurement of total hemoglobin, TritonX-100 was added to the samples to give a final concentration of 9 mMduring preincubation. Triton X-100 up to 18 mM does not interfere withhemoglobin absorbance measurements at 540 nm.RESULTS AND DISCUSSIONTable I shows that a two-fold decrease in ionic strength,reported to effect shape changes (discocyte to spherocyte)but not to exceed the critical hemolytic volume (CHV)(6), resulted in a significant activation of phospholipase A2toward PE and PC, with PC the preferred substrate.The action of phospholipase A2 toward erythrocytes asTABLE IACTION OF COBRA VENOM PHOSPHOLIPASE A2 TOWARD HUMAN ERYTHROCYTES AT VARIOUS OSMOTICSTRENGTHSTotalOsmotic Phospholipid (mol fraction) phospholipid Totalstrength(mosmol) PE LPE PC LPC PS SM hydrolysis hemolysis(%) (%)306 0.37 + 0.02 0.00 ± 0.00 0.31 ± 0.04 0.01 ± 0.00 0.14 ± 0.02 0.16 + 0.01 1 0188 0.38 + 0.04 0.02 + 0.00 0.27 ± 0.06 0.06 + 0.01 0.14 ± 0.01 0.14 ± 0.01 8 1108 0.31 + 0.01 0.10 ± 0.01 0.26 ± 0.01 0.02 + 0.00 0.15 ± 0.01 0.16 + 0.01 12 *The numbers quoted are the mean and error from the mean for duplicate determinations. Lyso PE (LPE) and Lyso PC (LPC) products are indicated;phosphatidylserine (PS) and sphingomyelin (SM) are not hydrolyzed.*Hemolysis prior to incubation experiments had occurred yielding red ghosts.166 BIOPHYS. J. © Biophysical Society * 0006-3495/82/01/166/03 $1.00Volume 37 January 1982 166-168kiILUa.IL.az0FU49IC.idS.-izla0U:a?iooEe80Z 60401080a604020v .aCh0Va.-wIL0z0FU49IC.Iw_IL 600a.A0Iz0 0.21.00.80.60.40.2004100so604020AlQ4 ci 08 1.0TRITON X- 100 (mM)FIGURE 1 The effect of Triton X-100 on erythrocyte hemolysis with (0)and without (0) enzyme present (bottom); phospholipase A2 hydrolysisof total PE and PC (middle); fraction of total phospholipid hydrolyzedwhich is PE (0) and PC (0) (top). Sphingomyelin is not a substrate ofthe enzyme and phosphatidylserine is not hydrolyzed significantly underthe assay conditions. At low concentrations of Triton X-100 hemolysis isenhanced by enzyme presence and hydrolysis.a function of Triton X-100 concentration is shown in Fig.1. Triton X-100 at 0.2 mM, which is just below its criticalmicelle concentration (c.m.c.), produces a large activationof the enzyme toward PE which cannot be accounted fortotally by hemolysis; 10% hemolysis should have exposed atotal of 0.07 Atmol combined PC and PE, yet four timesthis amount of lipid (0.28 ,umol) is hydrolyzed of whichover 90% is PE. Increasing surfactant concentrationfurther, which results in almost total hemolysis, did notlead to a further dramatic activation of the enzyme and PEremained the preferred substrate.In Fig. 2, melittin is observed to be lytic at allconcentrations tested and phospholipase A2 hydrolysisappears to parallel hemolysis. At extremely low concentra-tions of melittin enzyme hydrolysis of PE is preferred; butwith higher concentrations of the basic peptide the speci-ficity of the enzyme becomes about equal toward bothsubstrates, PE and PC.To the extent that hemolysis is an all-or-none phenome-non of hemoglobin release from erythrocytes beyond theCHV (6), under prehemolytic conditions the majority ofthe erythrocytes are intact and their outer surface isat0 20 30MELIflIN (PM)40 50FIGURE 2 The effect of melittin on erythrocyte hemolysis with (-) andwithout (0) enzyme present (bottom); phospholipase A2 hydrolysis oftotal PE plus PC (middle); fraction of total phospholipid hydrolyzedwhich is PE (-) and PC (0) (top). Sphingomyelin is not a substrate ofthe enzyme and phosphatidylserine is not hydrolyzed significantly underthe assay conditions. Hemolysis profiles for melittin show that enzymepresence does not result in further dectable hemolysis.available for probing by the phospholipase A2. The prelim-inary observations reported here raise the interesting possi-bility that a large part of the apparent activation ofphospholipase A2 hydrolysis is directed toward the outererythrocyte membrane surface, and results at least in partfrom prehemolytic subtle alterations in the outermembrane surface produced by lowering the ionic strengthor by introducing small amounts of Triton X-100. Theactivation of the enzyme toward PE when ionic strength isdecreased further, which results in hemolysis, and activa-tion at prehemolytic concentrations of the nonionic surfac-tant Triton X-100, suggest the intriguing possibility thatthese treatments perturb the membrane and lead to anincreased exposure of PE for enzyme hydrolysis at theouter surface of the membrane. Haest and Deuticke (7),using sulfhydryl reagents to perturb the erythrocytemembrane, also report increased exposure of PE to phos-pholipase A2 hydrolysis at the outer membrane surface.This work was supported by grant GM-20501 from the National Insti-tutes of Health.Receivedfor publication I May 1981.POSTER SUMMARIESla IL-WI i I I I. .-----.A_drI 0...I t I16771 1 IREFERENCES1. Adamich, M, and E. A. Dennis. 1978. Exploring the action andspecificity of cobra venom phospholipase A2 toward erythrocytes,ghost membranes, and lipid mixtures. J. Biol. Chem. 253:5121-5125.2. Adamich, M., and E. A. Dennis. 1979. Action of cobra venomphospholipase A2 toward lipids of erythrocyte membranes. InNormal and Abnormal Red Cell Membranes. C. Fox, editor. AlanR. Liss, Inc., New York. 515-521.3. Roberts, M. F., M. Adamich, R. J. Robson, and E. A. Dennis. 1979.Phospholipid activation of cobra venom phospholipase A2. 1. Lipid-lipid or lipid-enzyme interaction. Biochemistry. 18:3301-3308.4. Adamich, M., M. F. Roberts, and E. A. Dennis. 1979. Phospholipidactivation of cobra venom phospholipase A2. 2. Characterization ofthe phospholipid-enzyme interaction. Biochemistry. 18:3308-3314.5. Anderson, E. P., H. M. Kalckar, K. Kurahashi, and K. J. Isselbacher.1957. A specific enzymatic assay for the diagnosis of congenitalgalactosemia 1. The consumption test. J. Lab. Clin. Med. 50:469-477.6. Nathan, D. G., and S. B. Shohet. 1971. Erythrocyte ion transport,defects and hemolytic anemia: hydrocytosis and desicocytosis. InThe Red Cell Membrane. R. I. Weed, E. R. Jaffe, P. A. Miescher,editors. Grune and Stratton, New York. 384.7. Haest, W. W. M., and B. Deuticke. 1976. Possible relationshipbetween membrane proteins and phospholipid asymmetry in thehuman erythrocyte membrane. Biochim. Biophys. Acta. 436:353-365.SPECIFICITY IN PROTEIN-MEMBRANE ASSOCIATIONS:THE INTERACTION OF GANGLIOSIDES WITHESCHERICHIA COLI HEAT-LABILE ENTEROTOXIN ANDCHOLERAGENJ. C. OSBORNE, JR., J. Moss, P. H. FISHMAN AND S. NAKAYANational Heart, Lung, and Blood Institute, and National Institutes ofNeurological andCommunicative Disorders and Stroke, National Institutes ofHealth, Bethesda, Maryland 20205U.S.A.D. C. ROBERTSONUniversity ofKansas, Lawrence, Kansas 60645 U.S.A.Gangliosides, which are complex glycospingolipids, havebeen implicated in the receptor-mediated effects of numer-ous agents, including enterotoxins and glycoproteinhormones (6). The nonpolar portion of these compounds,consisting of an N-acyl fatty acid derivative of sphingo-sine, is believed to serve as an anchor in the plasmamembrane, whereas the carbohydrate moiety, which isexposed to the extracellular space, is believed to be respon-sible for receptor specificity. One of the more fully charac-terized ganglioside-protein interactions is that betweenganglioside GMI (galactosy-N-acetylgalactosaminyl-[N-acetyl-neuraminyl]-galactosylglucosylceramide) and theenterotoxin of Vibrio cholerae that is responsible for theclinical characteristics of cholera (3). Choleratoxin orcholeragen (CT) is composed of two types of subunits heldtogether by noncovalent interactions. The A component,which is believed to be composed of two polypeptide chainsjoined by a single disulfide bond, activates adenylatecyclase through a NAD-dependent ADP-ribosylationreaction (9). The B component, of which there are 4-6molecules in the intact toxin, is responsible for the specificinteraction between the toxin and GM, (3, 10). Thus theinitial event in the action of CT on cells is the binding ofthe B component to the oligosaccharide 'moiety of GM,.The remaining events are not known with certainty; the Alsubunit is currently believed to be inserted into themembrane bilayer (presumably after reduction of thedisulfide bond that links it to A2) and ultimately ADP-ribosylates a regulatory protein in the cyclase system.The effects of a heat-labile enterotoxin of Escherichiacoli (LT) on cells resemble in many ways those of CT. LTalso appears to be structurally similar to CT in that itcontains two types of subunits with molecular weightssimilar to those found for CT (1 1). The catalytic A subunitof LT is however believed to be a single polypeptide chain(5, 7) rather than a disulfide linked dimer as found for CT(1, 2, 3). In view of the similar structure and mode ofaction of these two enterotoxins we have investigated theinteraction between LT and GM,.RESULTS AND DISCUSSIONIncorporation of purified GM, into GM,-deficient C6glioma cells increased the subsequent binding of '251I-LT or'251I-CT. This increase was blocked for both toxins by priortreatment of GM,-treated cells with unlabeled CT. Treat-ment of the cells with GDla (N-acetylneuraminylgalac-tosyl-N-acetylgalatosaminyl-[N-acetylneuraminyl]-galac-tosylglucosylceramide, GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide) orGD b (galactosyl-N-acetylgalactosaminyl- [N-acetyl-neuraminyl-N-acetylneuraminyl] -galactosylglucosyl-168 BIOPHYS.J.©BiophysicalSociety * 0006-3495/82/01/168/02 $1.00Volume 37 January 1982 168-169

Phospholipase a(2) action on membrane surfaces: effect of ionic strength, triton x-100, and melittin.

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