Bacterial phospholipases C.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.     

Bacterial phospholipases C.

A variety of pathogenic bacteria produce phospholipases C, and since the discovery in 1944 that a bacterial toxin (Clostridium perfringens alpha-toxin) possessed an enzymatic activity, there has been considerable interest in this class of proteins. Initial speculation that all phospholipases C would have lethal properties has not been substantiated. Most of the characterized enzymes fall into one of four groups of structurally related proteins: the zinc-metallophospholipases C, the sphingomyelinases, the phosphatidylinositol-hydrolyzing enzymes, and the pseudomonad phospholipases C. The zinc-metallophospholipases C have been most intensively studied, and lethal toxins within this group possess an additional domain. The toxic phospholipases C can interact with eukaryotic cell membranes and hydrolyze phosphatidylcholine and sphingomyelin, leading to cell lysis. However, measurement of the cytolytic potential or lethality of phospholipases C may not accurately indicate their roles in the pathogenesis of disease. Subcytolytic concentrations of phospholipase C can perturb host cells by activating the arachidonic acid cascade or protein kinase C. Nonlethal phospholipases C, such as the Listeria monocytogenes PLC-A, appear to enhance the release of the organism from the host cell phagosome. Since some phospholipases C play important roles in the pathogenesis of disease, they could form components of vaccines. A greater understanding of the modes of action and structure-function relationships of phospholipases C will facilitate the interpretation of studies in which these enzymes are used as membrane probes and will enhance the use of these proteins as models for eukaryotic phospholipases C.     

Multiple phosphoinositide-specific phospholipases C in oat roots: characterization and partial purification

Phosphoinositide-specific phospholipases C are critical enzymes in the transduction of hormonal and environmental signals into animal cells. Several different isozymes of phospholipase C are known in mammalian systems which differ in their expression and regulation. Elucidation of the regulation of these phospholipases C has greatly advanced our understanding of the control of mammalian cell growth and development. Plant cells, too, contain phospholipases C specific for phosphoinositides, and there is evidence that they may be involved in plant cell responses to environmental stimuli. This paper reports that there are at least four variants of phosphoinositide-specific phospholipase C in the roots of oat seedlings, two cytosolic and two plasma membrane-associated. The two cytosolic and two plasma membrane variants can be separated on the basis of their affinity for binding to heparin. Both the cytosolic and the plasma membrane heparin-binding forms have apparent molecular weights of about 50–70 kDa by size exclusion chromatography. The two heparin-binding forms have been partially purified. The partially purified enzymes are activated by micromolar calcium and are specific for phosphorylated phosphoinositides; in their substrate specificities they resemble mammalian phospholipases C ɛ.     

Phospholipase activities of the P388D1 macrophage-like cell line

The murine macrophage (M phi) cell line, P388D1, was employed as a source of M phi phospholipases in order to characterize the enzymatic properties and subcellular localization of these enzymes because of their importance for prostaglandin biosynthesis. Phospholipase activity was assessed with dipalmitoylphosphatidylcholine (DPPC) as substrate. Phospholipases were characterized with respect to divalent cation dependence, pH optima, and localization in subcellular compartments using linear sucrose gradients. By these criteria a number of different phospholipases were identified. Most importantly, a single Ca2+-dependent activity with a pH optimum of 8.8 was identified in membrane-rich fractions (plasma membrane, mitochondria, and endoplasmic reticulum) and could be clearly separated from the remaining activities, which are Ca2+ independent and exhibit pH optima of 7.5, 5.1, and 4.2. The phospholipases with acidic pH optima may be associated with subcellular components containing lysosomal enzymes and both phospholipase A1 and phospholipase A2 activities are observed. In contrast, the phospholipase activity with a pH optimum of 7.5 sediments with the cytosolic proteins and is inhibited by 5 mM Ca2+. No significant phospholipase C activity was detected in assays performed with or without added Ca2+ at pH's 4.2, 5.1, 7.5, or 8.8 using DPPC as substrate. However, the P388D1 cells do contain a lysophospholipase that is at least 20 times more active than the phospholipase A activities identified. Its presence must be taken into account in evaluating the positional specificities and properties of the macrophage phospholipases.     

Preferential hydrolysis of peroxidized phospholipid by lysosomal phospholipase C

The susceptibility of partially peroxidized liposomes of 2-[1-14C] linoleoylphosphatidylethanolamine ([14C]PE) to hydrolysis by cellular phospholipases was examined. [14C]PE was peroxidized by exposure to air at 37 degrees C, resulting in the formation of more polar derivatives, as determined by thin-layer chromatographic analysis. Hydrolysis of these partially peroxidized liposomes by lysosomal phospholipase C associated with cardiac sarcoplasmic reticulum, and by rat liver lysosomal phospholipase C, was greater than hydrolysis of non-peroxidized liposomes. By contrast, hydrolysis of liposomes by purified human synovial fluid phospholipase A2 or bacterial phospholipase C was almost completely inhibited by partial peroxidation of PE. Lysosomal phospholipase C preferentially hydrolyzed the peroxidized component of the lipid substrate which had accumulated during autoxidation. The major product recovered under these conditions was 2-monoacylglycerol, indicating sequential degradation by phospholipase C and diacylglycerol lipase. Liposomes peroxidized at pH 7.0 were more susceptible to hydrolysis by lysosomal phospholipases C than were liposomes peroxidized at pH 5.0, in spite of greater production of polar lipid after peroxidation at pH 5.0. Sodium bisulfite, an antioxidant and an inhibitor of lysosomal phospholipases, prevented: (1) lipid autoxidation, (2) hydrolysis of both non-peroxidized and peroxidized liposomes by sarcoplasmic reticulum and (3) loss of lipid phosphorus from endogenous lipids when sarcoplasmic reticulum was incubated at pH 5.0. These studies show that lipid peroxidation may modulate the susceptibility of phospholipid to attack by specific phospholipases, and may therefore be an important determinant in membrane dysfunction during injury. Preservation of membrane structural and functional integrity by antioxidants may result from inhibition of lipid peroxidation, which in turn may modulate cellular phospholipase activity.     

Evidence for involvement of guanine nucleotide-binding regulatory proteins in the activation of phospholipases by hormones

Guanine nucleotide-binding regulatory proteins similar to Gs and Gi may be involved in the activation of phospholipases C and A2 by hormones and other ligands. The binding of hormones to receptors that activate phospholipase C is decreased by guanine nucleotides and these hormones also stimulate a high-affinity GTPase activity in cell membranes. Effects of hormones on phospholipase C activity in cell-free preparations are dependent on the presence of guanine nucleotides. In addition, fluoride and nonhydrolyzable GTP analogs activate phospholipases in a manner that can be blocked by GDP beta S. The putative guanine nucleotide-binding regulatory protein that appears to be involved in activation of phospholipase C is sensitive to pertussis toxin in some cells but not in others.     

Action of phospholipases on the phosphatidylcholine exchange protein from beef liver.

The phospholipases A2, C and D have been used to investigate the localization of phosphatidylcholine in the phosphatidylcholine exchange protein from beef liver. The rate of enzymatic hydrolysis of the protein-bound phosphatidylcholine was found to be very low. Addition of deoxycholate, isobutanol or dioxane to the native protein, under conditions where delipidation did not occur, greatly enhanced the hydrolytic action of the phospholipases. From these results it is concluded that phosphatidylcholine may be buried in the protein molecule.     

蛋白激酶C研究的最新进展

作为能使蛋白激酶C(PKC)活化的第二信使甘油二酯(DAG)不仅可由磷脂酰肌醇(PtdIns)水解产生,大量实验表明还可从磷脂酰胆碱(PC)水解而来,其中磷脂酶C(PLC)及磷脂酶D(PLD)参与了这一过程,磷脂酶A₂(PLA₂)的作用产物脂肪酸(FA)也能激活PKC.PKC至少有10种亚型,依据其活化方式可分三大类:典型PKC,新PKC和非典型PKC.PKC参与了基因表达的调控.     

Interaction of bilirubin with human erythrocyte membranes. Bilirubin binding to neuraminidase- and phospholipase-treated membranes.

Saturable bilirubin binding to human erythrocyte membranes was measured before and after digestion with neuraminidase and phospholipases. Neuraminidase-treated erythrocyte membranes did not show any change in their binding properties, indicating that gangliosides could be excluded as candidates for saturable bilirubin-binding sites on erythrocyte membranes. Although bilirubin-binding properties of the membranes did not change after phospholipase D digestion, either, phospholipase C treatment greatly enhanced bilirubin binding. Thus it is suggested that a negatively charged phosphoric acid moiety of phospholipids on the membrane surface may play a role to prevent a large amount of bilirubin from binding to the membranes. Further saturable bilirubin binding to inside-out sealed erythrocyte membrane vesicles showed values comparable with those of the right-side-out sealed membranes, suggesting that the bilirubin-binding sites may be distributed on both outer and inner surfaces of the membranes, or may exist in the membranes where bilirubin may be accessible from either side.     

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases.

1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50% of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80% of the sphingomyelin. 2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect. 3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved. 4. Due to its absolute requirement for Ca2+, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca2+ stimulates phospholipase A2 activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25% of the lecithin, half of the phosphatidylethanolamine and some 65% of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.     

An appraisal of the role of biliary phospholipases in the pathogenesis of gallstone disease

Human bile contains proteins that influence nucleation of cholesterol. Recently, it has been suggested that activity of phospholipases in bile may play a role in this process. To study the influence of phospholipase on nucleation we have determined the effect of phospholipases A2, C and D on the nucleation time of model bile. Phospholipase C decreased the nucleation time, whereas phospholipase A2 inhibited nucleation. The phospholipases were effective only at relatively high concentrations. Phospholipase D was strongly inhibited in model bile and probably only influenced the nucleation time by an aspecific protein effect. The cleavage products of the different phospholipases were determined in native bile samples of 14 cholesterol gallstone patients, 6 patients without stones and 4 patients with pigment stones. In all samples, choline, phosphorylcholine and free fatty acids (FFA) could be detected. However, there was no significant difference between the three groups of patients. The rate of production of choline, phosphorylcholine and FFA was measured in bile incubated at 37 degrees C. Again, there was no significant difference between the three groups of patients. We conclude that phospholipase activity in bile does not play an important role in the pathogenesis of gallstone disease.     

Effect of the state of the lipid phase of the membrane on the effectiveness of photochemical modification of erythrocyte acetylcholinesterase

Modification of the lipid phase structure of the erythrocyte membrane by phospholipases A2, C and D as well as the partial depletion of cholesterol was shown to be accompanied by the change of the acetylcholinesterase (AChE) UV-sensitivity. The ability of UV-light to change the catalytic properties (Km) of the membrane-bound AChE not observed for free AChE (constant value of Km) and known as the phenomenon of photochemical allotopy, is retained in the cholesterol depleted membranes and disappears after an enzymatic treatment of the membranes by phospholipases. The possible non-photochemical influence of the membrane lipid phase in response to UV-damage of membrane-bound AChE is discussed.     

Phospholipases may play multiple roles in anisotropic plant cell growth

Both the cortical microtubule cytoskeleton and cellulose microfibrils are important for the anisotropic growth of plant cells. Although the two systems interact, the details of this interaction are far from clear. It has been shown the inhibitors of phospholipase D, phospholipase A₂ and phospholipase C all cause disorganisation of the microtubule cytoskeleton. Since the phospholipases act on the plasma membrane, which links cortical microtubules to cellulose microfibrils in the cell wall, they may play a key role in the communication between the two structures. This communication may take various forms. Microtubule-linked phospholipase activity may cause the organisation of underlying cellulose microfibril liquid crystals. Alternatively, phospholipases may co-operate in the regulation of plasma membrane fluidity, affecting the movement of cellulose synthase complexes in the underlying plasma membrane. GPI-anchored proteins in the plasma membrane, which are cleaved by phospholipases, may possibly play a role.     

Effect of Retinoic Acid on the Ca2+-Independent Phospholipase A2 in Nuclei of LA-N-1 Neuroblastoma Cells

LA-N-1 neuroblastoma cell cultures contain Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and ethanolamine plasmalogens. These enzymes differ from each other in their molecular mass, substrate specificity, and kinetic properties. Subcellular distribution studies have indicated that the activity of these phospholipases is not only localized in the cytosol but also in non-nuclear membranes and in nuclei. The treatment of LA-N-1 neuroblastoma cell cultures with retinoic acid results in a marked stimulation of Ca²⁺-independent phospholipases A₂ hydrolyzing phosphatidylethanolamine and plasmenylethanolamine. The increase of the activities of both enzymes was first observed in nuclei followed by those present in the cytosol. No effect of retinoic acid on either phospholipase activity could be observed in non-nuclear membranes. The stimulation of these enzymes may be involved in the generation and regulation of arachidonic acid and its metabolites during differentiation.     

Amiodarone — an inhibitor of phospholipase activity: a comparative study of the inhibitory effects of amiodarone,chloroquine and chlorpromazine

Amiodarone, an antiarrhythmic drug, like chloroquine and chlorpromazine, is a tertiary amine with amphiphilic properties. Chloroquine and chlorpromazine are known inhibitors of phospholipases. All three drugs produce characteristic microcorneal deposits consistent with lysosomal accumulations of phospholipid. Similar lysosomal bodies were found in leukocytes of 15 patients on chronic amiodarone treatment as well as 3 patients each on chloroquine and chlorpromazine, suggestive of widespread systemic inhibition of lysosomal phospholipases. These lysosomal inclusions were similar in morphology, irrespective of the drug given, and were of four types: multilamellar, amorphous dense, amorphous light, or a combination of 2 or more of the preceding types. There was no simple relationship between the number of inclusion bodies per cell and the cumulative dose of amiodarone (r=0.02) or amiodarone serum levels (r=0.11). An in vitro assay was used to compare the effects of the three drugs on Ca²⁺-dependent phospholipase A and C activities. Phospholipase A₂ activity was inhibited in a dose-dependent fashion (1–8 mg/assay) by all three drugs in the order: chlorpromazine > amiodarone > chloroquine. The inhibitory effect on phospholipase C was more pronounced with all three drugs, producing almost total inhibition at 8 mg/assay. In a Ca²⁺-independent lysosomal phospholipase A system, amiodarone had a greater effect, producing 85% inhibition at 1.2 mg/assay. These observations suggest that amiodarone, like other cationic amphiphiles, induces a generalized phospholipidosis by inhibiting phospholipid catabolism. Its therapeutic and toxic effects may be due to its ability to modulate both Ca²⁺-dependent membrane phospholipases and Ca²⁺-independent acid phospholipases.     

Evidence for a phospholipid requirement in the specific binding of glucocorticoids to receptors of fibroblasts and thymic lymphocytes.

The specific steroid binding capacity of soluble preparations from mouse fibroblasts and rat thymic lymphocytes is inactivated by incubation with phospholipases. Receptor binding is drastically reduced by very low concentrations of boiled phospholipase A preparations from bee venom and snake venoms. The enzyme effect is calcium-dependent and is blocked by both phospholipid and a substrate analog that is a competitive inhibitor of phospholipase A. The specific binding capacity is also sensitive to digestion by phospholipase C. Two possible mechanisms are considered for the phospholipase A effect: (a) the receptor protein may be associated with a phospholipid component which is required for specific hormone binding; (b) phospholipase A may be producing detergent products that are indirectly inactivating the receptor. Examination of the effects of lysophosphatide on the receptor and assay of lipid phosphate in the receptor preparation do not support a mechanism based solely on detergent effects. Because phospholipase C, which does not produce detergent products, also inactivates the binding, we propose that the phospholipases may be digesting the phospholipid which is a requisite component of the glucocorticoid receptor.     

Glycosylphosphatidylinositol-anchored proteins: structure, function, and cleavage by phosphatidylinositol-specific phospholipase C.

A wide variety of proteins are tethered by a glycosylphosphatidylinositol (GPI) anchor to the extracellular face of eukaryotic plasma membranes, where they are involved in a number of functions ranging from enzymatic catalysis to adhesion. The exact function of the GPI anchor has been the subject of much speculation. It appears to act as an intracellular signal targeting proteins to the apical surface in polarized cells. GPI-anchored proteins are sorted into sphingolipid- and cholesterol-rich microdomains, known as lipid rafts, before transport to the membrane surface. Their localization in raft microdomains may explain the involvement of this class of proteins in signal transduction processes. Substantial evidence suggests that GPI-anchored proteins may interact closely with the bilayer surface, so that their functions may be modulated by the biophysical properties of the membrane. The presence of the anchor appears to impose conformational restraints, and its removal may alter the catalytic properties and structure of a GPI-anchored protein. Release of GPI-anchored proteins from the cell surface by specific phospholipases may play a key role in regulation of their surface expression and functional properties. Reconstitution of GPI-anchored proteins into bilayers of defined phospholipids provides a powerful tool with which to explore the interactions of these proteins with the membrane and investigate how bilayer properties modulate their structure, function, and cleavage by phospholipases.     

Stabilization of synaptic membranes with alpha-tocopherol against the damaging effect of phospholipases. Possible mechanism of the biological action of vitamin E

Using fluorescent and EPR spin probing techniques, the effects of phospholipases A2, C and D on rat brain synaptosomal membranes were investigated. It was shown that treatment of synaptosomal membranes with phospholipases A2, C and D results in their depolarization and increase of their surface negative charge. In case of phospholipases A2 and C, these changes are also accompanied by a decrease of the microviscosity of the synaptosomal membrane lipid bilayer. alpha-Tocopherol protects synaptosomal membranes against the damaging action of phospholipases. The stabilization of synaptosomes by vitamin E consists in the reconstitution of the transmembrane potential and in an increased microviscosity of phospholipase-treated membranes. The stabilizing effect of alpha-tocopherol is due to the binding of phospholipid hydrolysis products rather than to the inhibition of phospholipases. The observed stabilization of synaptosomal membranes by alpha-tocopherol is interpreted as a feasible mechanism of biological effects of vitamin E on biological membranes.     

Vanadate inhibits degradation of short-lived, but not of long-lived, proteins in L-132 human cells.

We have analysed the membrane anchorage of plasma-membrane 5'-nucleotidase, an ectoenzyme which can mediate binding to components of the extracellular matrix. We demonstrated that the purified enzyme obtained from chicken gizzard and a human pancreatic adenocarcinoma cell line were both completely transformed into a hydrophilic form by treatment with phospholipases C and D, cleaving glycosylphosphatidylinositol (GPI). These data indicate the presence of a glycolipid linker employed for membrane anchoring of the 5'-nucleotidase obtained from both sources. Incubation of plasma membranes under identical conditions revealed that about half of the AMPase activity was resistant to GPI-hydrolysing phospholipases. Investigation of the enzymic properties of purified chicken gizzard 5'-nucleotidase revealed only minor changes after removal of the phosphatidylinositol linker. However, cleavage of the membrane anchor resulted in an increased sensitivity towards inhibition by concanavalin A. After tissue fractionation, chicken gizzard 5'-nucleotidase could be obtained as either a membrane-bound or a soluble protein; the latter is suspected to be released from the plasma membrane by endogenous phospholipases. Higher-molecular-mass proteins immuno-cross-reactive with the purified chicken gizzard 5'-nucleotidase were detected as both soluble and membrane-bound forms.     

Interaction of aminoglycoside antibiotics with surface Asp and Glu residues of phosphatidylinositol-specific phospholipase C

The aminoglycoside antibiotics such as neomycin, gentamicin, kanamycin and streptomycin stimulated the purified enzyme phosphatidylinositol-specific phospholipases C from Bacillus thuringiensis at pH 5.5. The involvement of net positive charge of aminoglycoside antibiotics (AA) on phosphatidylinositol-specific phospholipases C activation was probed by modifying the carboxyl group of Asp and Glu present in the enzyme by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC). Intrinsic Trp fluorescence of EDAC modified and unmodified PI-PLC in the presence of AA confirmed the interaction of AA with side chain carboxyl group of aspartic and glutamic acid of the enzyme. Thus, the possible interaction of aminoglycoside antibiotics with phosphatidylinositol-specific phospholipases C is predicted to be mediated through the aspartic and glutamic acid residue(s) of the protein.