Adsorption of sphingomyelinase of Bacillus cereus onto erythrocyte membranes

Sphingomyelinase of Bacillus cereus proved to be specifically adsorbed onto mammalian erythrocyte membranes in the presence of either Ca2+ or Ca2+ plus Mg2+ in the order of sphingomyelin content; i.e., sheep, bovine greater than porcine greater than rat erythrocytes. No appreciable adsorption was observed in the presence of Mg2+ alone nor in the absence of divalent metal ions. The enzyme adsorption onto bovine erythrocytes was dependent upon the incubation temperature. By shifting the temperature from 37 to 0 degrees C, sphingomyelinase once adsorbed onto the surface of bovine erythrocytes was released into the supernatant. Ca2+ proved to be an essential factor for the enzyme adsorption: The addition of 1 mM Ca2+ enhanced the adsorptive process, but inhibited sphingomyelin hydrolysis and hot or hot-cold hemolysis of erythrocytes, while the addition of 1 mM Ca2+ plus 1 mM Mg2+ enhanced sphingomyelin breakdown and hemolysis as well as the enzyme adsorption. However, when the amount of sphingomyelin fell off to 0.2-0.7 nmol/ml or less by the action of sphingomyelinase, the enzyme once adsorbed was completely released from the surface of erythrocytes. The result indicates that the major binding site for sphingomyelinase is sphingomyelin. In the presence of 1 mM Mg2+ alone, the enzymatic hydrolysis of sphingomyelin and hemolysis proceeded whereas the enzyme adsorption was not encountered during 60 min incubation at 37 degrees C. The change in the molar ratio of Ca2+ to Mg2+ affected the enzyme adsorption and sphingomyelin breakdown; the higher Ca2+ enhanced the adsorption whereas the higher Mg2+ stimulated sphingomyelin hydrolysis.     

Hemolysis induced by Bacillus cereus sphingomyelinase

Bacillus cereus sphingomyelinase (Bc-SMase) induces hemolysis of sheep erythrocytes which contain large amounts of sphingomyelin. We investigated the mechanism of this hemolysis in comparison to that induced by Clostridium perfringens alpha-toxin. Pertussis toxin, a Gi-specific inhibitor, N-oleoylethernolamine, a ceramidase inhibitor, and dihydrosphingosine, a sphingosine kinase inhibitor, did not inhibit the hemolysis by Bc-SMase, but did inhibit that by alpha-toxin. Bc-SMase broadly bound to whole membranes, and alpha-toxin specifically bound to the detergent-resistant membrane fractions, lipid rafts. The level of ceramide production induced by Bc-SMase in sheep erythrocytes was 6- to 15-fold that induced by alpha-toxin, when the extent of the hemolysis by Bc-SMase was the same as that by the toxin. However, the level of ceramide production induced by Bc-SMase in SM-liposomes was equal to that triggered by the toxin, when the carboxyl fluorescein-release from liposomes induced by Bc-SMase was the same as that induced by alpha-toxin. Confocal laser microscopy showed that treatment of the cells with Bc-SMase resulted in the formation of ceramide-rich domains. A photobleaching analysis suggested that treatment of the cells with Bc-SMase leads to a reduction in membrane fluidity. These results show that Bc-SMase-induced hemolysis of sheep erythrocytes is related to the formation of interface between ceramide-rich domains and ceramide-poor domains through production of ceramide from SM.     

Increase in osmotic fragility of bovine erythrocytes induced by bacterial phospholipases C

Bovine erythrocytes were treated with each of three bacterial phospholipases C; phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens, sphingomyelinase C (SMase) of Bacillus cereus and phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis. An increase in osmotic fragility was detected by means of a coil planet centrifugation (CPC) apparatus (Biomedical Systems Co., Tokyo) after the treatment with these enzymes. The peak of hemolysis normally observed in the untreated erythrocytes at the range between 50 and 100 mOsM shifted to 160 to 200 mOsM with the progress of sphingomyelin hydrolysis by phospholipase C of C. perfringens. Sphingomyelinase C of B. cereus showed two different effects on bovine erythrocytes: In the absence of divalent cations or in the presence of Ca2+ alone, the peak of hemolysis shifted to the region from 130 to 160 mOsM, without appreciable hydrolysis of sphingomyelin, while in the presence of Mg2+ or Mg2+ plus Ca2+, the peak of hemolysis further shifted to the region from 160 to 200 mOsM with the hydrolysis of sphingomyelin. Abrupt shift in osmotic fragility to a much higher region around 250 mOsM was produced by treatment with increasing amounts of phosphatidylinositol-specific phospholipase C. In this case, a significant amount of acetylcholinesterase was released from the erythrocyte membrane without hot or hot-cold hemolysis. The mechanism of alteration of osmotic fragility of bovine erythrocytes by treatment with phospholipases C seems to differ from case to case, depending upon the specific action of each enzyme toward the membrane phospholipids.     

The relationship of membrane lipids to species specific hemolysis by hemolytic factors from Stomoxys calcitrans (L.) (Diptera: Muscidae)

Posterior-midgut homogenate from female stable flies prepared at 12 h after feeding hemolyzed erythrocytes from 6 different mammalian species more readily than homogenate prepared at 22 h. A significant correlation was obtained between the per cent sphingomyelin content of the erythrocyte membrane and the time required for lysis by the 12 h homogenate. Erythrocytes with low sphingomyelin content were more sensitive to lysis than cells with high sphingomyelin. No such correlation exists for hemolysis by 22 h homogenate. Mean corpuscular volume and osmotic fragilities of erythrocytes were not related to hemolysis either by 12 or 22 h homogenate. Determination of phospholipase C and sphingomyelinase activities showed that the hydrolysis rate of phospholipase C in homogenates prepared at 12–14 h was almost twice as much as sphingomyelinase activity. Whereas hydrolysis rates in 22–24 h homogenate were not different and markedly reduced compared to the 12–14 h homogenate. The times required for erythrocyte hemolysis related to the phospholipase C and sphingomyelinase activity profiles suggests that these enzyme activities participate in the in vitro hemolysis of red blood cells. Bovine and human erythrocytes change their biconcave contour into a spiculated spherical shape when they are exposed to midgut homogenate. This shape change is interpreted as a detergent induced modification of the red cell membrane which renders the erythrocytes more vulnerable to hemolysis.     

The action of sphingomyelinase from Bacillus cereus on ATP-depleted bovine erythrocyte membranes and different lipid composition of liposomes

The presence of cholesterol or phosphatidylethanolamine in sphingomyelin liposomes enhanced 2- to 10-fold the breakdown of sphingomyelin by sphingomyelinase from Bacillus cereus. On the other hand, the presence of phosphatidylcholine was either without effect or slightly stimulative at a higher molar ratio of phosphatidylcholine to sphingomyelin (3/1). In the bovine erythrocytes and their ghosts, the increase by 40-50% or the decrease by 10-23% in membranous cholesterol brought about acceleration or deceleration of enzymatic degradation of sphingomyelin by 50 or 40-50%, respectively. The depletion of ATP (less than 0.9 mg ATP/100 ml packed erythrocytes) enhanced K+ leakage from, and hot hemolysis (lysis without cold shock) of, bovine erythrocytes but decelerated the breakdown of sphingomyelin and hot-cold hemolysis (lysis induced by ice-cold shock to sphingomyelinase-treated erythrocytes), either in the presence of 1 mM MgCl2 alone or in the presence of 1 mM MgCl2 and 1 mM CaCl2. Also, ATP depletion enhanced the adsorption of sphingomyelinase onto bovine erythrocyte membranes in the presence of 1 mM CaCl2 up to 81% of total activity, without appreciable K+ leakage and hot or hot-cold hemolysis. These results suggest that the presence of cholesterol or phosphatidylethanolamine in biomembranes makes the membranes more susceptible to the attack of sphingomyelinase from B. cereus and that the segregation of lipids and proteins in the erythrocyte membranes by ATP depletion causes the deceleration of sphingomyelin hydrolysis despite the enhanced enzyme adsorption onto the erythrocyte membranes.     

Changes in phosoholipid susceptibility toward phospholipases induced by ATP depletion in avian and amphibian erythrocyte membranes.

About half of the sphingomyelin content of fresh and ATP-depleted chicken erythrocytes is hydrolysed by sphingomyelinase. Removal of spingomyelin exposes the rest of the membrane phospholipids to hydrolysis by phospholipase C only in ATP-depleted but not in fresh cells. Addition of both sphinogomyelinase and phospholipase C to ATP-depleted cells causes about 60-70 percent hydrolysis of the total phospholipids accompanied by extensive (90 percent) hemolysis. The phospholipids of toad erythrocytes are partially available to phospholipase C activity in fresh cells (17-25 percent hydrolysis) without prior sphingomyelinase treatment. However, in ATP-depleted toad cells phospholipase C hydrolyses 66 percent of phospholipids and causes extensive lysis. Treatment of either fresh or ATP-depleted toad erythrocytes by sphingomyelinase together with phospholipase C induces hydrolysis of most of the phospholipds with complete lysis. Restoration of ATP to ATP-depleted cells endows them with resistance to the attack of phospholipase C. The correlation between changes in ATP level and membrane organization as revealed by increased susceptibility toward phospholipases is discussed.     

Enzymatic oxidation of membrane cholesterol in relation to lysis of sheep erythrocytes by corynebacterial enzymes

Synergistic hemolysis of sheep erythrocytes brought about by the combined action of Corynebacterium ovis (C. pseudotuberculosis) and Corynebacterium equi depends upon the extracellular sphingomyelin-specific phospholipase D of the former species and a partially characterized agent(s) of the latter. Fractionation of the culture supernatant of C. equi revealed a cholesterol oxidase which was purified to near homogeneity by gel filtration and isoelectric focusing. The enzyme was isoelectric at pH 9–10 and had a molecular weight of 61,000. Sheep erythrocytes pretreated with purified sphingomyelinase D of C. ovis were hemolyzed by incubation with C. equi cholesterol oxidase or by the same enzyme from Brevibacterium sp. Lipid analysis revealed complete conversion of membrane cholesterol to cholest-4-en-3-one, the product of cholesterol oxidase action. Cells not pretreated with sphingomyelinase D did not undergo cholesterol oxidation or hemolysis when treated with cholesterol oxidase. Studies with crude culture supernatant of C. equi confirmed the presence of a phospholipase active in hydrolyzing ceramide phosphate generated in the erythrocyte membrane by C. ovis sphingomyelinase. Ceramide thus produced in the membrane is known to make the cells labile to hemolysis. There are, therefore, at least two mechanisms underlying synergistic hemolysis by these coryne-bacteria.     

Leakage-free membrane fusion induced by the hydrolytic activity of PlcHR(2), a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

PlcHR(2) is the paradigm member of a novel phospholipase C/phosphatase superfamily, with members in a variety of bacterial species. This paper describes the phospholipase C and sphingomyelinase activities of PlcHR(2) when the substrate is in the form of large unilamellar vesicles, and the subsequent effects of lipid hydrolysis on vesicle and bilayer stability, including vesicle fusion. PlcHR(2) cleaves phosphatidylcholine and sphingomyelin at equal rates, but is inactive on phospholipids that lack choline head groups. Calcium in the millimolar range does not modify in any significant way the hydrolytic activity of PlcHR(2) on choline-containing phospholipids. The catalytic activity of the enzyme induces vesicle fusion, as demonstrated by the concomitant observation of intervesicular total lipid mixing, inner monolayer-lipid mixing, and aqueous contents mixing. No release of vesicular contents is detected under these conditions. The presence of phosphatidylserine in the vesicle composition does not modify significantly PlcHR(2)-induced liposome aggregation, as long as Ca(2+) is present, but completely abolishes fusion, even in the presence of the cation. Each of the various enzyme-induced phenomena have their characteristic latency periods, that increase in the order lipid hydrolysis相似文献   

Suppression of high-pressure-induced hemolysis of human erythrocytes by preincubation at 49 degrees C.

When human erythrocytes were preincubated at 37-52 degrees C under atmospheric pressure before exposure to a pressure of 200 MPa at 37 degrees C, the value of hemolysis was constant (about 43%) up to 45 degrees C but became minimal at 49 degrees C. The results from anti-spectrin antibody-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamide-labeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49 degrees C. The vesicles released at 200 MPa by 49 degrees C-preincubated erythrocytes were smaller than those released by the treatment at 49 degrees C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45 degrees C, and the vesicles released from 49 degrees C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45 degrees C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49 degrees C, compression produces smaller vesicles, so that the hemolysis is suppressed.     

Abnormal transbilayer mobility of phosphatidylcholine in hereditary pyropoikilocytosis reflects the increased heat sensitivity of the membrane skeleton

We determined whether the membrane defect in hereditary pyropoikilocytosis (HPP) is associated with thermally induced changes in the lipid bilayer, the stability of which was probed by the rate of translocation of phosphatidylcholine (PC) over the two leaflets. [14C]PC was incorporated into the outer leaflet of the lipid bilayer of the intact erythrocytes using a PC-specific phospholipid exchange protein. The transbilayer equilibration of this PC was determined by measuring the time-dependent changes in its accessibility to exogenous phospholipase A2. The rate of transbilayer equilibration of PC was increased in HPP cells at 37 degrees C when compared to normal erythrocytes (rate constants, 0.07 +/- 0.02 and 0.03 +/- 0.01 h-1, respectively). A further dramatic increase in PC transbilayer equilibration was noted in HPP cells incubated at 44 degrees C (rate constant, 0.15 +/- 0.02 h-1). A similar marked acceleration in transbilayer movement of PC was also seen in normal erythrocytes when incubated at 46 degrees C (rate constant, 0.13 +/- 0.03 h-1). Despite the enhanced transbilayer mobility of PC in HPP cells when compared to normal erythrocytes, no major alteration in the asymmetric distribution could be observed when probed with phospholipase A2. Since changes in transbilayer mobility of PC and cell morphology occur in HPP cells at lower temperature than in normal red cells, it may be concluded that the enhanced thermal sensitivity of spectrin is the major factor responsible for these changes. Our results therefore support the view that the structural integrity of the skeletal network is essential for stabilization of the lipid bilayer of the red cell membrane.     

Utilization of membranous lipid substrates by membranous enzymes: activation of the latent sphingomyelinase of hen erythrocyte membrane

Previous studies demonstrated that hen erythrocytes have an inoperative, latent sphingomyelinase which is activated when the cells are hemolyzed in a hypotonic medium. Within minutes after hemolysis about 60-80% of the sphingomyelin (SPM) of the RBC "ghost" membrane was hydrolyzed. In this paper, expression of sphingomyelinase activity was further investigated. The percentage of total SPM hydrolyzed depended on the volume of the hypotonic hemolyzing buffer. Thus, suspending the erythrocytes in 4 vol of the buffer resulted in clumping of the hemolyzed "ghosts" and no hydrolysis of SPM. In comparison, suspension in 19 vol of the hypotonic buffer showed no clumping and sphingomyelinase activity was fully expressed. But centrifugation of the latter or, alternatively, addition of concanavalin A induced clumping and elimination of sphingomyelinase activity. Hen RBC could also be hemolyzed in an isotonic medium in the presence of Triton X-100, mellitin, halothane, and phospholipase C. Activation of the latent sphingomyelinase occurred at concentrations of these reagents which caused cell lysis. Hen RBC were dispersed in an isotonic medium containing glutaraldehyde (0.1%) or formaldehyde (10%). This rendered the cells resistant to hemolysis, even when subsequently dispersed in a hypotonic medium or water. But incubation of the "fixed" cells in a hypotonic or isotonic medium activated the enzyme, resulting in hydrolysis of 60% of the cellular SPM. In contrast, when glutaraldehyde was included in the hypotonic buffer, hemolysis occurred but sphingomyelinase activity was eliminated.     

A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).     

Endovesiculation of human erythrocytes exposed to sphingomyelinase C: a possible explanation for the enzyme-resistant pool of sphingomyelin

When human erythrocytes are treated with Staphylococcus aureus sphingomyelinase C at 37 degrees C they become susceptible to cold lysis and appear to endovesiculate. Endovesiculation has been confirmed by showing that in parallel with sphingomyelin breakdown, the cells accumulate [3H]inulin or [14C]sucrose (without losing intracellular K+) and also experience a loss of cell-surface acetylcholinesterase activity into a latent intracellular pool which can be revealed by treatment with detergent. On the basis of these observations it can be calculated that endovesicles account for about 2-4% of cell volume and about 25% of total cell surface. Pretreatment of cells with bee venom phospholipase A2 completely blocked sphingomyelinase-induced endovesiculation but this effect was related to a concomitant decrease in sphingomyelin breakdown which was reduced by about 90%. These results indicate that the pool of sphingomyelin which is not susceptible to attack by sphingomyelinase C (about 15% of total sphingomyelin) may be resistant because of membrane internalisation and not because it originally resides in the inner leaflet of the plasma membrane.     

The role of sphingomyelin in regulating phase coexistence in complex lipid model membranes: competition between ceramide and cholesterol

The structure, thermotropic phase behavior, dynamic motion and order parameters of bilayer dispersions of egg phosphatidylcholine, egg sphingomyelin, egg ceramide and cholesterol have been determined. The coexistence of gel, liquid-ordered and liquid-disordered structure has been determined by peak fitting analysis of synchrotron X-ray powder patterns. Order parameters and extent of distribution of 16-doxyl-stearic acid spin probe between ordered and disordered environments has been estimated by ESR spectral simulation methods. The presence of ceramide in proportions up to 20 mol% in phosphatidylcholine is characterized by gel-fluid phase coexistence at temperatures up to 46 degrees C depending on the amount of ceramide. Cholesterol tends to destabilize the ceramide-rich domains formed in phosphatidylcholine while sphingomyelin, by formation of stable complexes with ceramide, tends to stabilize these domains. The stability of sphingomyelin-ceramide complexes is evident from the persistence of highly ordered structure probed by ESR spectroscopy and appearance of a sharp wide-angle X-ray reflection at temperatures higher than the gel-fluid transition of ceramide alone in egg phosphatidylcholine bilayers. The competition between ceramide and cholesterol for interaction with sphingomyelin is discussed in terms of control of lipid-mediated signaling pathways by sphingomyelinase and phospholipase A2.     

Cholesterol modulation of sphingomyelinase activity at physiological temperatures

Bacillus cereus sphingomyelinase activity was assayed on large unilamellar vesicles composed of sphingomyelin (SM)/cholesterol (Ch) mixtures at varying proportions. Natural (egg) SM was used with a gel–fluid transition temperature at ca. 40 °C. When the enzyme was assayed at 37 °C, the activity on pure SM was exceedingly low, but a small increase was observed as soon as some Ch was added, and a large enhancement of activity occurred with Ch proportions above 25 mol%. The data were interpreted in terms of sphingomyelinase activity being higher in the cholesterol-induced liquid-ordered phase than in the gel phase. The abrupt increase in activity above 25 mol% Ch would occur as a result of a change in domain connectivity, when the Ch-rich liquid-ordered domains coalesced. In equimolar SM/Ch mixtures, that were in the liquid-ordered state in a wide range of temperatures, sphingomyelinase activity was virtually constant in the 30–70 °C range. The results demonstrate that at the mammalian and bird physiological temperatures Ch modulates sphingomyelinase activity, and that this can occur precisely because most SM have a gel–fluid transition temperature above the physiological temperature range. In addition, Ch activation of sphingomyelinase and the strong affinity of Ch for SM allow the rapid, localised and self-contained production of the metabolic signal ceramide in specific microdomains (rafts).     

Clostridium perfringens alpha-toxin activates the sphingomyelin metabolism system in sheep erythrocytes

Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism. Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine. We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes. Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P). N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells. Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate. These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis. S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells. Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM. In addition, incubation of C. botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P. These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.     

Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine

Sphingomyelin (SM) and phosphatidylcholine (PC) are two major phospholipids on plasma lipoproteins. Their concentration is classically measured by lipid extraction, thin-layer chromatography, and phosphate determination on separated SM or PC spots. Here, we describe two rapid, specific, and sensitive enzymatic measurements for both phospholipids. Plasma was incubated with bacterial sphingomyelinase (for SM measurement) or bacterial PC-specific phospholipase D (for PC measurement), alkaline phosphatase, choline oxidase, peroxidase, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline, and 4-aminoantipyrine for 45 min. A blue dye, with an optimal absorption at 595 nm, was generated. PC levels did not influence SM measurement and vice versa. The linear range for the SM measurement was 0.5-5 microg, and that for PC was 2.5-20 microg. The mean percentage recovery was 98.0 +/- 5.2% for SM and 96.6 +/- 3.8% for PC. The interassay coefficient of variation of the assay was 1.7 +/- 0.05% for SM and 3.1 +/- 0.13% for PC. These two new methods are amenable to automation and can be adapted for large-scale, high-throughput assays.     

A novel class of microbial phosphocholine-specific phospholipases C

In this report we describe the 1,500-fold purification and characterization of the haemolytic phospholipase C (PLC) of Pseudomonas aeruginosa, the paradigm member of a novel PLC/phosphatase superfamily. Members include proteins from Mycobacterium tuberculosis, Bordetella spp., Francisella tularensis and Burkholderia pseudomallei. Purification involved overexpression of the plcHR1,2 operon, ion exchange chromatography and native preparative polyacrylamide gel electrophoresis. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry confirmed the presence of two proteins in the purified sample with sizes of 17,117.2 Da (PlcR2) and 78,417 Da (PlcH). Additionally, liquid chromatography electrospray mass spectrometry (LCMS) revealed that PlcH and PlcR2 are at a stoichiometry of 1 : 1. Western blot analysis demonstrated that the enzyme purifies as a heterodimeric complex, PlcHR2. PlcHR2 is only active on choline-containing phospholipids. It is equally active on phosphatidylcholine (PC) and sphingomyelin (SM) and is able to hydrolyse plasmenylcholine phospholipids (plasmalogens). Neither PlcHR2 nor the M. tuberculosis homologues are inhibited by D609 a widely used, competitive inhibitor of the Bacillus cereus PLC. PlcH, PlcR2, and the PlcHR2 complex bind calcium. While calcium has no detectable effect on enzymatic activity, it inhibits the haemolytic activity of PlcHR2. In addition to being required for the secretion of PlcH, the chaperone PlcR2 affects both the enzymatic and haemolytic properties of PlcH. Inclusive in these data is the conclusion that the members of this PC-PLC and phosphatase family possess a novel mechanism for the recognition and hydrolysis of their respective substrates.     

Sphingomyelinase generation of ceramide promotes clustering of nanoscale domains in supported bilayer membranes

The effects of ceramide incorporation in supported bilayers prepared from ternary lipid mixtures which have small nanoscale domains have been examined using atomic force and fluorescence microscopy. Both direct ceramide incorporation in vesicles used to prepare the supported bilayers and enzymatic hydrolysis of SM by sphingomyelinase were compared for membranes prepared from 5:5:1 DOPC/sphingomyelin/cholesterol mixtures. Both methods of ceramide incorporation resulted in enlargement of the initial small ordered domains. However, enzymatic ceramide generation led to a much more pronounced restructuring of the bilayer to give large clusters of domains with adjacent areas of a lower phase. The individual domains were heterogeneous with two distinct heights, the highest of which is assigned to a ceramide-rich phase which is hypothesized to occur via ceramide flip-flop to the lower leaflet with formation of a raised domain due to negative membrane curvature. A combination of AFM and fluorescence showed that the bilayer restructuring starts rapidly after enzyme addition, with formation of large clusters of domains at sites of high enzyme activity. The clustering of domains is accompanied by redistribution of fluid phase to the periphery of the domain clusters and there is a continued slow evolution of the bilayer over a period of an hour or more after the enzyme is removed. The relevance of the observed clustering of small nanoscale domains to the postulated coalescence of raft domains to form large signaling platforms is discussed.     

Effects of temperature and bovine serum albumin on lysis of erythrocytes induced by dilauroylglycerophosphocholine and didecanoylglycerophosphocholine.

The effects of the incubation temperature and bovine serum albumin on hemolysis induced by short-chain phosphatidylcholine were examined. The rate of hemolysis of human, monkey, rabbit, and rat erythrocytes by dilauroylglycerophosphocholine showed biphasic temperature-dependence: hemolysis was rapid at 5-10 degrees C and above 40 degrees C, but slow at around 25 degrees C. In contrast, the rate of lysis of cow, calf, sheep, pig, cat, and dog erythrocytes did not show biphasic temperature-dependence, but increased progressively with increase in the incubation temperature. Bovine serum albumin increased the hemolysis of human erythrocytes induced by dilauroylglycerophosphocholine or didecanoylglycerophosphocholine: it shortened the lag time of lysis and reduced the amount of phosphatidylcholine required for lysis. A shift-down of the incubation temperature from 40 to below 10 degrees C also shortened the lag time of lysis of human erythrocytes induced by dilauroylglycerophosphocholine and reduced the amount of phosphatidylcholine required for lysis.