Modification of the tetrodotoxin receptor in Electrophorus electricus by phospholipase A2

The effects of phospholipase A₂ treatment on the tetrodotoxin receptors in Electrophorus electricus was studied. (1) The binding of [³H]tetrodotoxin to electroplaque membranes was substantially reduced by treatment of the membranes with low concentrations of phospholipase A₂ from a number of sources, including bee venom, Vipera russelli and Crotalus adamanteus and by $\text{β-}\text{bungarotoxin}$. (2) Phospholipase A₂ from bee venom and from C. adamanteus both caused extensive hydrolysis of electroplaque membrane phospholipids although the substrate specificity differed. Analysis of the phospholipid classes hydrolyzed revealed a striking correlation between loss of toxin binding and hydrolysis of phosphatidylethanolamine but not of phosphatidylserine. (3) The loss of toxin binding could be partially reversed by treatment of the membranes with bovine serum albumin, conditions which are known to remove hydrolysis products from the membrane. (4) Equilibrium binding studies on the effects of phospholipase A₂ treatment on [³H]tetrodotoxin binding showed that the reduction reflected loss of binding sites and not a change in affinity. (5) These results are interpreted in terms of multiple equilibrium states of the tetrodotoxin-receptors with conformations determined by the phospholipid environment.     

Possible relationship between membrane proteins and phospholipid asymmetry in the human erythrocyte membrane

After incubation of human erythrocytes at 37 °C in the absence of glucose (A) for 24 h, (B) for 4 h with 8 mM hexanol or (C) for 3 h with SH reagents, phosphatidylethanolamine becomes partly susceptible to hydrolysis by phospholipase A₂ from Naja naja. The presence of glucose during the pretreatments suppresses this effect, except in the case of SH reagents that inhibit glycolysis. After incubation with tetrathionate, up to 45% of the phosphatidylethanolamine is degraded by the enzyme, an amount considerably in excess of the 20% attacked in fresh erythrocytes.Pancreatic phospholipase A₂, an enzyme unable to hydrolyse the phospholipids of intact erythrocytes, partially degrades phosphatidylcholine and phosphatidylethanolamine of erythrocytes pretreated with hexanol or SH reagents. Reagents capable of oxidizing SH groups to disulfides (tetrathionate, $\text{o-}\text{iodosobenzoate}$ and hydroquinone) even render susceptible to pancreatic phospholipase A₂ phosphatidylserine, a phospholipid supposed to be entirely located in the inner lipid layer of the membrane. Alkylating or acylating SH reagents have no such effect. It is postulated that disulfide bond formation between membrane protein SH groups leads to an alteration in protein-phospholipid interactions and consequently induces a reorientation of phospholipids between the inner and the outer membrane lipid layer.     

Pancreatic phospholipase A2 is not regulated by calmodulin

It was previously suggested [Wong, P.Y.-K and Cheung, W.Y. (1979) Biochem. Biophys. Res. Comm. $\text{90}$, 473–480] that the Ca²⁺ activation of phospholipase A₂ is mediated by the calcium binding protein calmodulin. In the present study phospholipase A₂ from pig pancreas was shown to be absolutely Ca²⁺ dependent but the enzyme was not stimulated by exogenous calmodulin and no endogenous calmodulin was found in the preparation. The enzyme was inhibited in the absence of calmodulin by several drugs (trifluoperazine, mepacrine, promethazine and propranolol) which are known to bind to calmodulin. A kinetic analysis indicated that trifluoperazine competitively inhibited phospholipase A₂, probably by interacting with phospholipid substrate.     

Characterization of phospholipase A2 and acyltransferase activities in squid (loligo pealei) axoplasm: comparison with enzyme activities in other neural tissues, axolemma and axoplasmic subfractions

Phospholipase A₂ and acyltransferase were assayed and characterized in pure axoplasm and neural tissues of squid. Intracellular phospholipase A₂ activity was highest in giant fiber lobe and axoplasm, followed by homogenates from retinal fibers, optic lobe and fin nerve. In most preparations, exogenous calcium (5 mM) caused a slight stimulation of activity. EGTA (2 mM) was somewhat inhibitory, indicating that low levels of endogenous calcium may be required for optimum activity. Phospholipase A₂ was inhibited by 0.1 mM p-bromophenacylbromide, and was completely inactivated following heating.The level of acylCoA: lysophosphatidylcholine acyltransferase activity was higher in axoplasm and giant fiber lobe than in other neural tissues of the squid. K_m (apparent) and V_max (apparent) for oleoyl-CoA and lysophosphatidylcholine were quite similar for axoplasm and giant fiber lobe enzyme preparations. Acyltransferase activity was inactivated by heat treatment, and greatly inhibited by 0.2 mM p-chloromercuribenzoate, and to a lesser extent by 20 mM N-ethylmaleimide.Phospholipase A₂ activity was present in fractions enriched in axolemmal membranes (separated from squid retinal fibers and garfish olfactory nerve) from both tissues, and it was also highly concentrated in vesicles derived from squid axoplasm. In all three preparations, phospholipase A₂ activity was stimulated by Ca⁺⁺ (5 mM) and inhibited by EGTA (2 mM). In addition, axoplasmic cytosol (114,000 g supernatant) retained a substantial portion of a Ca⁺⁺-independent phospholipase A₂, active in the presence of 2 mM EGTA. Acyltransferase activity was present at high content in both axolemma membrane rich fractions, and among subaxoplasmic fractions and axoplasmic vesicles.     

Further characterization of Ca2+-activated phospholipase A2 in cat adrenal cortex

When cat adrenocortical cells were incubated with exogenous phospholipid substrate (autoclaved $\text{E.}$$\text{coli}$) in the presence of corticotropin, there was a Ca²⁺-dependent increase in phospholipid breakdown activity, suggesting that a hormone-stimulated phospholipase is localized to the plasma membrane. Phospholipase activity in a particulate fraction from lysed cells at neutral pH was a function of the Ca²⁺ concentration. The addition of increasing Ca²⁺ concentrations to a subcellular fraction of lysed cells which had been prelabelled with [¹⁴C]arachidonic acid produced graded increases in fatty acid release. A depletion of label from phosphatidylcholine was observed, as well as a marked increase in radioactivity associated with phosphatidylethanolamine. The subcellular fraction of cells prelabelled with [¹⁴C]palmitic acid failed to release fatty acid in response to Ca²⁺, although a loss of label from phosphatidylcholine and a modest gain in label by phosphatidylethanolamine was demonstrable. A Ca²⁺-activated deacylation-reacylation reaction preferentially involving phosphatidylethanolamine was evident in cortical cells prelabelled with archidonic acid; whereas, other Ca²⁺-stimulated lipolytic reactions also appeared to be operative in cells prelabelled with either arachidonic or palmitic acid. The Ca²⁺-dependent mobilization of arachidonic acid from an endogenous phospholipid pool lends additional support to the idea that Ca²⁺-mediated activation of phospholipase A₂ participates in the control of adrenocortical activity. However, since Ca²⁺ also stimulated arachidonic acid liberation from cortical triglycerides, these lipid moieties may also contribute to the observed effects of Ca²⁺ on fatty acid release.     

Arachidonic acid, bradykinin and phospholipase A2 modify both prolactin binding capacity and fluidity of mouse hepatic membranes

The objective of this study was to determine if arachidonic acid, a precursor of prostaglandin synthesis, bradykinin, a decapeptide known to stimulate membrane phospholipid methylation, arachidonic acid release and prostacyclin synthesis, and enzyme phospholipase A₂, capable of liberating arachidonic acid, alter the fluidity of hepatic membranes which could in turn modify the functionality of prolactin receptors. Liver homogenates of adult C₃H female mice incubated at 28°C for various times with 1–20 μg/ml arachidonic acid, 1–100 μg/ml bradykinin or 0.26–0.00026 U/ml phospholipase A₂ provided the 100,000 × g membrane pellets for subsequent ovine prolactin binding and membrane fluidity studies. Membrane microviscosity was determined by fluorescence polarization techniques using the lipid probe 1,6 diphenylhexatriene. Arachidonic acid, bradykinin and phospholipase A₂ stimulated specific oPRL binding, in a dose-related fashion, with maximum increases of 73%, 21% and 46%, at 4 μg/ml arachidonic acid, 5 μg/ml bradykinin and 0.026 U/ml PLA₂, respectively. This induction, occurring within 30 min of incubation, was found to be due to an increase in the number of receptor sites. Under the same conditions, arachidonic acid, bradykinin and PLA₂ induced 22%, 16%, and 18% decreases in membrane microviscosity, respectively. These data suggest that prostaglandin synthesis modifying agents may modulate the number of prolactin receptors $\text{in vivo}$ by changing the lipid fluidity of the target cell membranes by either of their known effects: arachidonic acid release from the phospholipid matrix, synthesizing appropriate prostaglandins at correct concentration or methylation of membrane phospholipids.     

Phospholipase A2 inactivation of microsomal 17β-hydroxysteroid oxidoreductase: Rates of phospholipid hydrolysis and enzyme inactivation,effects of hydrolysis products and properties of the phospholipase A2-treated enzyme

Exposure of guinea pig liver microsomes to phospholipase A₂ resulted in the nearly complete loss of 17β-hydroxy-steroid oxidoreductase (17β-HSD) activity, the time course of which correlated with phospholipid hydrolysis and lysolecithin formation. Lysolecithin and unsaturated fatty acids added to microsomes also inactivated 17β-HSD indicating that they may contribute to the inactivation by phospholipase A₂.If exposure to lysolecithin and fatty acids was minimized by including serum albumin in the reaction mixture, phospholipids were rapidly hydrolyzed; but in this case the extent of 17β-HSD inactivation was less and the rate of loss was significantly slower. The data suggest that phospholipid hydrolysis $\text{per se}$ results in a destabilization of 17β-HSD resulting in the subsequent activity loss.The inactivation of 17β-HSD by lysolecithin and fatty acids has not been reported previously and is suggestive of a possible control mechanism $\text{in vivo}$.     

THE DIFFERENTIATION OF PHOSPHOLIPASE A1 AND A2 IN RAT AND HUMAN NERVOUS TISSUES

—Lipid-free extracts of rat and human brain have been prepared and shown to contain phospholipase A₁ and A₂ activities and a lysophospholipase. The phospholipase Aj activity has pH optima of 4·2 and 4·6 in rat and human brain, respectively; it can be partially purified and isolated in high yields by dialysing the extracts at low pH. The purified preparations hydrolyse the ester bond at the 1-position in lecithin, phosphatidyl-ethanolamine and phosphatidylserine, but have little or no action on triglyceride or cholesterol ester. An assay system for the enzyme is described. Phospholipase A₂ activity is optimal at pH 5·5 in rat brain extracts and at pH 5·0 in extracts of human brain. The phospholipase A₂ activity of human cerebral cortex is largely unaffected by heating extracts at 70°C for 5 min, whereas this treatment substantially inactivates phospholipase A₁ and completely destroys lysophospholipase. Phospholipase A₁ is widely distributed in both grey and white matter of human brain and is also present in peripheral nerve. Phospholipase A₂ activity is lower than A₁ in all regions of the CNS examined so far, and is absent from peripheral nerve. Neither enzyme appears to require Ca²⁺ but both are inhibited by di-isopropylfluorophosphate (DFP, 2 × 10^?6 m) and thus differ from phospholipase A of pancreas. These studies confirm that the phospholipase A₁ and A₂ activities in brain are due to separate enzymes.     

Crotoxin effects on Torpedo californica cholinergic excitable vesicles and the role of its phospholipase A activity

The phospholipolytic neurotoxin from $\text{Crotalus}\text{durissus}\text{terrificus}$, crotoxin, is able to produce a dose- and time-dependent block of carbachol-stimulated ²²Na efflux from pre-loaded $\text{Torpedo}\text{californica}$ excitable vesicles. The blocking activity is dependent on calcium and is abolished by chemical modification with p-bromophenacyl bromide. The isolated basic subunit, crotoxin B, produces an identical block, whereas the isolated acidic subunit, crotoxin A, has no detectable effect. Neither crotoxin nor crotoxin B antagonizes the binding of $\text{[}{}^{125}\text{I]-α-bungarotoxin}$ to purified acetylcholine receptor, although, at high concentrations, they antagonize its binding to acetylcholine receptor-rich membrane fragments. Certain phospholipase A₂ enzymes and the fatty acid products of their digestion can mimic the crotoxin action. It is therefore suggested that, although considered a pre-synaptic neurotoxin, crotoxin can have $\text{in}\text{vitro}$ post-synaptic effects, possibly mediated by its endogeneous phospholipase A₂ activity.     

The release of phospholipase A2 from aggregated platelets and stimulated macrophages of sheep

Phospholipase A₂ release from platelets and macrophages was assayed by means of a modified, rapid bioassay based on the hydrolysis of $\text{E. coli}$ membranes. Enzyme activity was found in the supernatants of both washed, aggregated platelets and concanavalin A stimulated macrophages from afferent lymph of sheep. Phospholipase A₂ may be related to the vasoactive moiety secreted by antigen or mitogen stimulated macrophages.     

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4-yl)amino]-caproyl]phosphatidylcholine (C₆-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A₂ (PA₂) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C₆-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca⁺⁺ was required for PA₂ catalysis. Identification of the products of hydrolysis showed cleavage at the $\text{sn}$-1 and $\text{sn}$-2 positions for LpL and PA₂, respectively. For PA₂, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.     

Phospholipase A inhibition of acetylcholine receptor function in Torpedo californica membrane vesicles

A protein isolated from $\text{Naja naja siamensis}$ venom on the basis of its phospholipase A activity inhibits acetylcholine receptor function in post-synaptic membrane vesicles from $\text{Torpedo californica}$. Specifically, the phospholipase A prevents the large increase in sodium efflux that can normally be induced by carbamylcholine, a receptor agonist. The phospholipase A inhibition shows the following properties: 1) it occurs at concentrations 50 times lower than the concentrations required for inhibition by α-neurotoxins; 2) the phospholipase A has no effect on the binding properties of the receptor; 3) the inhibition is abolished by removal of calcium ions; and 4) some phospholipid hydrolysis accompanies inhibition. It is suggested that the phospholipase A acts enzymatically to uncouple ligand binding from ion permeability in the receptor containing membrane vesicles.     

Work in progress. Occurence of phospholipase A1 and A2 in human decidua

Phospholipase A₂, an enzyme which may regulate the formation of polyunsaturated fatty acids utilized for prostaglandin synthesis, was found to have significant higher activity in decidual than in myometrial tissue. The major part of phospholipase A₂ in the decidua had an acid pH optimum, which indicates that most of the enzyme is stored in the lysosomes of this tissue. These findings, together with previous observations, lend further support to the view that lysosomal phospholipase A₂ released within decidual cells might be a trigger of abortion and parturition.     

Effect of phospholipid methylation on calcium transport and (Ca2+ + Mg2+)-ATPase activity in kidney cortex basolateral membranes

Basolateral membranes isolated from hog kidney cortex, enriched 12- to 15-fold in (Na⁺ + K⁺)-ATPase activity, were 80% oriented inside-out as determined by assay of oubain-sensitive (Na⁺ + K⁺)-ATPase activity before and after opening of the membrane vesicle preparation with a mixture of deoxycholate and EDTA. In these membrane preparations 80% of total phosphatidylethanolamine was accessible to trinitrophenylation by trinitrobenzenesulfonic acid at 4°C, while at 37°C all of phosphatidylethanolamine fraction was chemically modified. Phospholipase C treatment resulted in hydrolysis of 80% phosphatidylethanolamine, 40% phosphatidylcholine and 35% of phosphatidylserine. Sphingomyelinase treatment resulted in 20% hydrolysis of sphingomyelin, presumably derived from right-side-out oriented vesicles. Results indicate that phosphatidylethanolamine is oriented exclusively on the outer leaflet of the lipid bilayer of inside-out oriented vesicles. Methylation of phospholipids in basolateral membranes with $\text{S-}\text{adenosyl}\text{[methyl-}{}^3\text{H}\text{]}\text{methionine}$ resulted in the three successive methylation of ethanolamine moiety of phosphatidylethanolamine to phosphatidylcholine. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for $\text{S-}\text{adenosylmethionine}$ was 1·10^?4 M with an optimum pH 9.0 for the formation of all three methyl derivatives. Mg²⁺ was without any effect between pH 5 and 10. Basolateral membranes incubated in the presence of methyl donor, $\text{S-}\text{adenosylmethionine}$, exhibited increased (12–15%) (Ca²⁺ + Mg²⁺)-ATPase activity and increased ATP-dependent uptake of calcium. ATP-dependent calcium uptake in these vesicles was insensitive to oligomycin and ouabain but was abolished completely by 50 μM vanadate. The increase in ATP-dependent calcium uptake was due to an increase in $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ and not due to a change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for Ca²⁺. Preincubation of membranes with $\text{S-}\text{adenosylhomocysteine}$, a methyltransferase inhibitor, abolished the stimulatory effect of phospholipid methylation on calcium uptake. Phospholipid methylation at both low and high pH did not result in a change in bulk membrane fluidity as determined by the fluorescence polarization of diphenylhexatriene. These results suggest that phospholipid methylation may regulate transepithelial calcium flux in vivo.     

The use of a phospholipase A-less Escherichia coli mutant to establish the action of granulocyte phospholipase A on bacterial phospholipids during killing by a highly purified granulocyte fraction

Phospholipase A₂ present in a highly purified, potently bactericidal, fraction from rabbit granulocytes produces net bacterial phospholipid degradation during killing of a phospholipase A-less strain of Escherichia coli. In the wild-type parent strain phospholipid breakdown is caused not only by the action of phospholipase A₂ but also by phospholipase A₁, indicating activation of the most prominent phospholipase of E. coli. This activation occurs as soon as the bacteria are exposed to the granulocyte fraction. Phospholipid breakdown by both phospholipases A is dose dependent but reaches a plateau after 30–60 min and at higher concentrations of the fraction.Phospholipid degradation is accompanied in both strains by an increase in permeability to actinomycin D that is also dose dependent. Even though net hydrolysis of phospholipids is greater in the parent strain than in the mutant, the increase in permeability is the same in the two strains.The addition of 0.04 M Mg²⁺, after the effects on phospholipids and permeability have become manifest, initiates in both strains the restoration of insensitivity to actinomycin D, the net resynthesis of phospholipids, and the disappearance of monoacylphosphatides and the partial disappearance of free fatty acids that had accumulated. Loss of ability to multiply is not reversed by Mg²⁺ in either strain. Less than 5 μg of granulocyte fraction causes loss of viability of from 90 to 99% of $\text{1 × 10}{}^8$ microorganisms of both strains. However, at lower concentrations the parent strain is considerably more sensitive to the bactericidal effect of the granulocyte fraction than the mutant strain.     

Cerebral microvessel phospholipase A2 activity in senescent mouse

Phospholipase A₂ activity was measured in cerebral microvessels isolated from 5 to 6 month (young adult) and 21 to 24 month (aged adult) old mice. Radiolabeled 1-stearoyl-2-[1-¹⁴C]arachidonyl choline phosphoglyceride was used as the enzyme substrate, and enzyme activity determined at pH 8 and pH 9. Activity in older animals was significantly less than in younger animals at both pH's. With choline phosphoglyceride as a substrate, phospholipase A₂ activity was predominantly Ca²⁺-dependent, although a small, but measurable Ca²⁺-independent component was present. Negligible production of diacylglycerol indicated little or no phospholipase C activity. These findings indicate that activity of a phospholipase A₂, which utilizes choline phosphoglyceride as a substrate, is affected by the aging process. Moreover, a change in PLA₂ activity would result in altered metabolism of specific phosphoglycerides and turnover of fatty acids at the sn-2 position in cerebral microvessels.     

Isolation and characteristics of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Phospholipase A₂ was purified from the pyloric ceca of the starfish Asterina pectinifera. The final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 20,000. The optimum pH and temperature of the enzyme were at around pH 9.0 and 50°C, respectively, and the activity was enhanced by sodium deoxycholate and 1 mM or higher concentration of Ca²⁺. The enzyme had no fatty acid specificity. Starfish phospholipase A₂ hydrolyzed phosphatidylcholine more effectively than phosphatidylethanolamine.     

Effect of purified phospholipases on the binding of tetrodotoxin to axon plasma membrane

Summary The role of phospholipids in the binding of [³H] tetrodotoxin to garfish olfactory nerve axon plasma membrane was studied by the use of purified phospholipases. Treatment of the membranes with low concentrations of either phospholipase A₂ (Crotalus adamanteus andNaja naja) or phospholipase C (Bacillus cereus andClostridium perfringens) resulted in a marked reduction in tetrodotoxin binding activity. A 90% reduction in the activity occurred with about 45% hydrolysis of membrane phospholipids by phospholipase A₂, and with phospholipase C the lipid hydrolysis was about 60–70% for a 70–80% reduction in the binding activity. Phospholipase C fromB. cereus andCl. perfringens had similar inhibitory effects. Bovine serum albumin protected the tetrodotoxin binding activity of the membrane from the inhibitory effect of phospholipase A₂ but not from that of phospholipase C. In the presence of albumin about 25% of the membrane phospholipids remained unhydrolyzed by phospholipase A₂. It is suggested that these unhydrolyzed phospholipids are in a physical state different from the rest of the membrane phospholipids and that these include the phospholipids which are directly related to the tetrodotoxin binding component. It is concluded that phospholipids form an integral part of the tetrodotoxin binding component of the axon membrane and that the phospholipase-caused inhibition of the binding activity is due to effects resulting from alteration of the phospholipid components.     

Cobra venom phospholipase A2 immobilized to porocus glass beads.

Phospholipase A₂ (EC 3.1.1.4) from cobra venom (Naja naja naja) has been covalently immobilized to aryl amine porous glass beads by diazo coupling. The attachment of the enzyme to the glass beads is apparently through tyrosine. The activity of the immobilized enzyme toward phospholipid substrate has been monitored using the Triton X-100/phospholipid mixed micelle assay system. The activity of the immobilized phospholipase A₂ toward phosphatidylcholine is about 160 μmol min^?1 ml^?1 of glass beads, and the specific activity is about 13 μmol min^?1 mg^?1 of protein in this assay system. The pH rate profile and apparent pK_a in 10 mm Ca²⁺ of the immobilized enzyme parallels that of the soluble enzyme. The substrate specificity of the immobilized enzyme toward individual phospholipid species in mixed micelles is phosphatidylcholine ? phosphatidylethanolamine. In binary lipid mixtures in mixed micelles containing phosphatidylcholine and phosphatidylethanolamine together, a reversal in specificity is observed, and phosphatidylethanolamine is the preferred substrate. This unusual specificity reversal in binary mixtures is also observed for the soluble enzyme. The activity of the immobilized enzyme toward phospholipid inserted in mixed micelles is the same as toward a synthetic phospholipid which forms monomers, while a 20-fold decrease in activity toward monomeric substrate is observed for the soluble enzyme. The immobilized enzyme is stable at temperatures of 90 °C as is the soluble enzyme. However, p-bromphenacyl bromide, a reagent which inactivates the soluble enzyme, does not inactivate the immobilized enzyme. The immobilized enzyme can be stored frozen for several months and is reusable. The mechanism of action of immobilized phospholipase A₂ from cobra venom and the potential usefullness of the bound enzyme as a probe for phospholipids in surfaces of membranes is considered.