Multiple forms of porcine pancreatic phospholipase A2: Isolation and specificity

Porcine pancreatic phospholipase A₂ was purified from commercial pancreatin by a method involving heat denaturation, trichloroacetic acid precipitation, and DEAE-cellulose chromatography. Assaying the eluate of the chromatography step by a new titrimetric method using vegetable lecithin-albumin emulsion as the substrate, several species of phospholipase A were found. Some of these went undetected when the conventional egg yolk emulsion assay was used. Two phospholipases A₂ were isolated in a homogeneous form and shown to have similar chemical and physical properties. Catalytic specificity of the two enzymes differs remarkably toward lecithins in different emulsified states.     

Evidence of phospholipase A2 in the sea urchin egg: Its possible involvement in the cortical granule reaction

Fertilization of the sea urchin egg involves an exocytotic event known as the cortical granule reaction (CGR). In many cell systems, phospholipase A₂ is implicated in regulation of the secretory event. Indirect evidence suggests that phospholipase A₂ mediates the CGR; however, there has been no direct demonstration of phospholipase A₂ activity in the sea urchin egg. We report here evidence of phospholipase A₂ activity in egg homogenate of the sea urchin Lytechinus pictus. The enzyme was calcium-dependent and had a pH optimum near the intracellular pH of the unfertilized egg. Neither exogenous calmodulin nor trifluoperazine had any apparent effect on enzyme activity. Quinacrine, a phospholipase A₂ inhibitor, blocked the enzyme activity in the egg homogenate. In intact eggs, quinacrine blocked the CGR in a dose-dependent, egg-concentration-dependent manner. The inhibitory effect of quinacrine on the CGR could not be overcome by the phospholipase A₂ activator melittin or by the calcium ionophore A23187. Quinacrine did not inhibit sperm-egg binding or sperm incorporation. These results lend further support to the hypothesis that phospholipase A₂ is involved in the CGR.     

Isolation and preliminary characterisation of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix)

Two toxic phospholipases A have been isolated from the venom of the Malayan cobra (Naja naja sputatrix). The phospholipases A were purified by successive ion-change chromatography on SP-Sephadex C-25, Sephadex G-75 gel filtration chromatography and successive Bio-Rex 70 ion-exchange chromatography. The purified toxic phospholipases A were homogeneous electrophoretically. They were designated as sputatrix phospholipase A-I and sputatrix phospholipase A-II. Positional specificity studies showed that they belong to the A₂-type phospholipase A. The medium lethal dose 50% (LD₅₀) values of the two phospholipases A are 0.27 and 0.28 μg/g, respectively, by intravenous injection and 1.05 and 1.00 μg./g, respectively, by intraperitoneal injection. The molecular weights of the two enzymes are 14 000 as determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Amino acid composition of sputatrix phospholipase A-I differs from sputatrix phospholipase A-II only by having one extra amino acid: a glutamic acid. Amino acid compositions of the two enzymes are also similar to those of other cobra venom phospholipases A.     

Purification of plant calmodulin by fluphenazine-Sepharose affinity chromatography.

The calcium-dependent binding of phenothiazine drugs to calmodulin (Levin, R. M. and Weiss, B. (1977) Mol. Pharmacol. $\text{13}$, 690–697) has been utilized to develop a rapid purification procedure for calmodulin based on fluphenazine-Sepharose affinity chromatography. Calmodulin from plant, a fungus, porcine brain and the coelenterate, $\text{Renilla}\text{reniformis}$, were easily purified by the calcium-dependent binding of calmodulin to fluphenazine-Sepharose.     

Calmodulin stimulates human platelet phospholipase A2.

Calmodulin is a ubiquitous Ca²⁺-binding protein, mediating the effect of Ca²⁺ on many enzyme systems and cellular reactions. Phospholipase A₂ (phosphatide-2-acyl-hydrolase, EC 3.1.1.4) which governs the level of arachidonic acid in human platelets, requires Ca²⁺ for maximum activity. Results presented herein suggest that the stimulation of phospholipase A₂ by Ca²⁺ is also mediated through calmodulin. This finding adds to the growing list of enzymes whose activities are regulated by calmodulin.     

Discrimination between the regioisomeric 1,2- and 1,3-diacylglycerophosphocholines by phospholipases

The artificial 1,3-diacyl-glycero-2-phosphocholines (1,3-PCs), which form similar aggregate structures as the naturally occurring 1,2-diacyl-sn-glycero-3-phosphocholines (1,2-PCs), were tested as substrates for different classes of phospholipases such as phospholipase A₂ (PLA₂) from porcine pancreas, bee and snake venom, and Arabidopsis thaliana, phospholipase C (PLC) from Bacillus cereus, and phospholipase D (PLD) from cabbage and Streptomyces species. The regioisomers of the natural phospholipids were shown to bind to all investigated phospholipases with an affinity similar to the corresponding naturally occurring phospholipids, however their hydrolysis was reduced to different degrees (PLA₂s and PLC) or even abolished (PLDs belonging to the PLD superfamily). The results are in accordance with binding models obtained by docking the substrates to the crystal structures or homology models of the phospholipases.     

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography

Intracellular phospholipase A₂ was purified to homogenity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A₂ from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A₂ examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A₂ from other sources.     

Action of phospholipase A2 and phospholipase C on Escherichia coli

The action of exogenous phospholipases on Escherichia coli has been examined. Cells harvested in late log phase were found to be completely resistant to the action of phospholipases A₂ and C. Treatment of cells with Tris and EDTA was required to make the phospholipids in the cell accessible to these phospholipases. Phospholipase A₂ hydrolyzed mainly phosphatidylethanolamine and phosphatidylglycerol, whereas phospholipase C preferentially degraded phosphatidylethanolamine.During the EDTA treatment, an endogenous phospholipase A₁ or a lysophospholipase (or both) was unmasked which caused the formation of free fatty acids in experiments in which no phospholipase was added and which degraded some of the lysophospholipids formed by phospholipase A₂.The cells were rapidly killed by the successive Tris-EDTA-phospholipase treatment, but no cell disintegration was observed.     

Calcium-dependent affinity chromatography of calmodulin on an immobilized phenothiazine.

The reversible, calcium-dependent binding of a calmodulin to phenothiazines has been demonstrated using an immobilized chlorpromazine analog. Calmodulin has been purified from crude extracts of bovine brain utilizing calcium-dependent binding to phenothiazine-Sepharose 4B as an initial affinity-based chromatographic procedure. Chromatography of a crude extract of bovine brain, prepared under non-denaturing conditions, yielded calmodulin contaminated with several other minor EGTA-elutable components. These components were removed by calcium-dependent affinity chromatography on calmodulin-Sepharose 4B and ion-exchange chromatography.     

The effect of calmodulin and chlorpromazine on the activity of phospholipases A2 from various sources

The effects of calmodulin and chlorpromazine on purified phospholipase A2 preparations from snake venoms: cobra (Naja naja oxiana), echis (Ehis multisquamatus) and Agkistrodon halys halys, as well as on phospholipases A2 from rat liver mitochondria and human platelets were studied. It was shown that within the concentration range of 1-5 microM calmodulin stimulates the phospholipase activity. Chlorpromazine inhibits the activity of these enzymes, the degree of inhibition being different for various phospholipases. Calmodulin was shown to interact with the phospholipases in the absence of exogenous Ca2+. The results obtained indicate that all phospholipases tested are calmodulin-dependent enzymes.     

Purification and characterization of a protein inhibitor of calcium-dependent proteases from rat liver

Soluble extracts of rat liver contain a protein inhibitor of calcium-dependent proteases. The inhibitor has an apparent M_r = 250,000 and is separated from the calcium-dependent proteases by gel-filtration chromatography in the presence of EGTA. The inhibitor has been purified by affinity chromatography using a calcium-dependent protease covalently linked to Affi-Gel 15. The inhibitor specifically binds to this affinity resin in a calcium-dependent manner and elutes in the presence of EDTA or EGTA. The purified inhibitor appears as a single protein with M_r = 125,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Presumably it is a dimer under nondenaturing conditions. The inhibitor inhibits each of two calcium-dependent proteases from rat liver and from other tissues and species. However, it has no effect on any other protease tested.     

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.     

High affinity binding of the mastoparans by calmodulin

Calmodulin exhibits high affinity, calcium-dependent binding of the mastoparans — a group of cytoactive tetradecapeptides. The dissociation constants for the peptide-calmodulin complexes determined in 0.20 N KCl, 1.0 mM CaCl₂, pH 7.3 are ～0.3 nM for mastoparan, ～0.9 nM for mastoparan X, and ～3.5 nM for $\text{Polistes}$ mastoparan. The dissociation constant for the mastoparan-calmodulin complex is the smallest known for any calmodulin binding protein or peptide, suggesting that some type of peptide-calmodulin interaction could be physiologically significant.     

A neurotoxic phospholipase a(2) impairs yeast amphiphysin activity and reduces endocytosis

Background

Presynaptically neurotoxic phospholipases A₂ inhibit synaptic vesicle recycling through endocytosis.

Principal Findings

Here we provide insight into the action of a presynaptically neurotoxic phospholipase A₂ ammodytoxin A (AtxA) on clathrin-dependent endocytosis in budding yeast. AtxA caused changes in the dynamics of vesicle formation and scission from the plasma membrane in a phospholipase activity dependent manner. Our data, based on synthetic dosage lethality screen and the analysis of the dynamics of sites of endocytosis, indicate that AtxA impairs the activity of amphiphysin.

Conclusions

We identified amphiphysin and endocytosis as the target of AtxA intracellular activity. We propose that AtxA reduces endocytosis following a mechanism of action which includes both a specific protein–protein interaction and enzymatic activity, and which is applicable to yeast and mammalian cells. Knowing how neurotoxic phospholipases A₂ work can open new ways to regulate endocytosis.     

An affinity ligand for phospholipase A2 purification

An alkyl ether analog of phosphatidylcholine was synthesized and used as a ligand to purify acid-extracted phospholipase A₂ from bovine ileum smooth muscle by affinity chromatography in the presence of cholate. This ligand contains a primary amino group at the ω-position of the acyl chain in position 1 and so permits direct covalent coupling with the ester group of Affi-Gel-10. An endogenous membrane bound phospholipase A₂ has been purified 32-fold in a good yield (70%) employing this ligand in an affinity chromatography step.     

Action of phospholipase A2 of rabbit neuronal and glial cells on 1,2-diacyl-, 2-acyl-1-alk-1′-enyl-, and 2-acyl-1-alkyl-glycerophosphatides

Pronounced differences in the phospholipase A₂ activities were found in neurons and glia, the enzyme activity being two- to threefold higher in neurons than in glial cells. Both phospholipases A₂ hydrolyzed the 1,2-diacylglycerophosphatides more rapidly than the acylalkyl and acylalkenyl compounds. Choline plasmalogen and the corresponding alkyl derivative were cleaved at similar rates by the phospholipase A₂ from both glia and neurons. There was a tendency by the neuronal phospholipase A₂ to release arachidonic acid faster than linolenic acid from both phosphatidylcholine and ethanolamine, while arachidonic acid was removed less actively from phosphatidylethanolamine by the glial enzyme. The glial phospholipase A₂ showed a lag period of 10 or 20 min. Norepinephrine, injected into the lateral ventricle of the rabbit brain, stimulated the hydrolysis of the various 1,2-diacyl-, acylalkyl-, and acylalkenyl-glycerophosphatides by the phospholipase A₂ from both glia and neurons.     

Properties of receptors for neurotoxic phospholipases A2 in different tissues

A radioiodinated derivative of OS₂ (¹²⁵I–OS₂), a neurotoxic monochain phospholipase A₂ isolated from taipan venom, was previously found to bind to a specific brain membrane receptor with very high affinity.¹²⁵I–OS₂ is now used to identify the properties of neurotoxic phospholipase receptors in other tissues. Heart, skeletal muscle, kidney, lung, liver, pancreas, and smooth muscle membranes also contain high-affinity binding sites for toxic phospholipases A₂. In most tissues, two different types of receptor sites have been characterized for¹²⁵I–OS₂ with K_d1 and K_d2 values in the 1–5 pM and the 10–50 pM range respectively. Whereas all receptors are similar in the different tissues in terms of their affinity for¹²⁵I–OS₂, maximal binding site capacities were very different, varying from 1.4 pmol/mg of protein in brain to 0.01 pmol/mg of protein in pancreaas. In brain, heart, and skeletal muscle, receptor densities vary with in vivo development. Affinity labeling experiments have identified the subunit composition of OS₂ receptors and indicated that these receptors do not have identical structures in the different tissues. Binding competition studies with OS₂ and other toxic phospholipases showed tissue-dependent pharmacological profiles. All these results taken together suggest the existence of a family of receptors for neurotoxic phospholipases.The abbreviations used are PLA₂ phospholipase A₂ - DSS suberic acid bis-N-hydroxysuccinimide ester - EDTA ethylenediaminetetraacetic acid - Hepes 4-(2-hydroxyethyl)1-piperazine ethanesulfonic acid - SDS sodium dodecyl sulfate - OS₁ Oxyuranus scutellatus scutellatus toxin 1 - OS₂ Oxyuranus scutellatus scutellatus toxin 2 Special issue dedicated to Dr. Lawrence Austin.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^–9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^?9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

Inhibition by Neurotoxic Phospholipases A2 of Synaptosomal Uptake of γ-Aminobutyric Acid

A comparison has been made of the abilities of several neurotoxic and nontoxic phospholipases A₂ from snake venoms to inhibit the intake of γ-aminobutyric acid into synaptosomes from rat cerebral cortex. The neurotoxic phospholipases A₂ inhibited GABA uptake more than the nontoxic enzymes did. However, there was a poor correlation between the measured specific enzyme activity of a phospholipase A₂ and its ability to inhibit the uptake of GABA.