The dependence of the effects of calcium ions on enzymatic hydrolysis of phospholipids on the physical state of the substrate]

The role of calcium ions in the phospholipid hydrolysis by phospholipase D was studied. It was shown that the enzyme does not split egg lecithine in the absence of Ca2+. In the presence of Ca2+ the reaction occurs via different routes, depending on the type of initiation of the reaction. The optimal concentrations of Ca2+ necessary for activation of phospholipase D are different in the systems activated by various treatments (organic solvents, detergents and solid adsorbents). Optimal concentrations of Ca2+ for the hydrolysis and methanolysis catalyzed by phospholipase D are also different. It was found that the need for Ca2+ and their optimal concentrations are determined by the state of phospholipids at the substrate phase. The data suggest that the enzymatic hydrolysis may occur in the absence of Ca2+. Thus, Ca2+-induced activation is merely an alternative pathway of catalytically active conformation of lypolytic enzymes.     

Phospholipase activity in rat liver mitochondria studied by the use of endogenous substrates

The hydrolysis of endogenous phosphatidyl ethanolamine and lecithin in rat liver mitochondria has been studied by using mitochondria from rats injected with ethanolamine-1,2-(14)C or choline-1,2-(14)C. A phospholipase A-like enzyme has been demonstrated, which catalyzes the hydrolysis of one fatty acid ester linkage in phosphatidyl ethanolamine and lecithin. Phosphatidyl ethanolamine is hydrolyzed in preference to lecithin and the main reaction products are free fatty acids and lysophosphatidyl ethanolamine. The further breakdown of lysophospholipids appears to be limited in mitochondria, which indicates that lysophospholipase activity is mainly located extramitochondrially. The enzymic system is greatly stimulated by calcium ions, and also slightly by magnesium ions, while EDTA inhibits it almost completely. These findings are discussed in relation to previous observations on the effect of calcium and of EDTA on the functions of mitochondria. The possible function of the mitochondrial phospholipase for the formation of phospholipids with special fatty acids at the alpha- and -position is discussed.     

Reaction of Local Anesthetics with Phospholipids : A possible chemical basis for anesthesia

Local anesthetics (LA) have been found to interact with phospholipids and lipids extracted from nerve and muscle. This reaction is demonstrated by: (a) Inhibition by LA of phospholipid (and tissue lipid) facilitated transport of calcium from a methanol: water phase into chloroform. This action is dependent upon the cationic form of the LA. (b) LA increase the electrical resistance of "membranes" prepared by impregnating Millipore filters with cephalin:cholesterol or tissue lipid extracts and bathed with NaCl or KCl solutions. (c) LA coagulate aqueous dispersions of cephalin, phosphatidyl serine, phosphatidyl ethanolamine, and inositide, an action shared by calcium. The order of potency in coagulating cephalin sols is tetracaine > calcium > butacaine > procaine. Na⁺ and K⁺ do not coagulate phospholipid dispersions at 0.1 M concentration and antagonize the effect of Ca²⁺. (d) LA produce a marked fall in the pH of cephalin sols equivalent to that produced by calcium, (e) Ca²⁺ and LA form 1:2 molar complexes with phospholipids probably by ion-ion and ion-induced polar type of binding at the phosphate groups of the lipid. It is suggested that such reactions with cell membrane phospholipids may underlie inhibitory effects of LA on cellular ion fluxes and provide a chemical basis for anesthetic action.     

A new kinetic approach for studying phospholipase C (Clostridium perfringens alpha toxin) activity on phospholipid monolayers

The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers. A two-step reaction was used. Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface. With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate. It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized. No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates. By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role. The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate.     

Regulatory aspects of mitochondrial phospholipase A2 from rat liver: effects of proteins, phospholipids and calcium ions

This paper deals with the search for specific inhibitors or activators of the mitochondrial phospholipase A2. Convincing evidence for the existence of proteins in the mitochondrial or cytosolic fraction that function as specific regulators of this enzyme was not obtained. The enzymatic activity appeared to be inhibited at low substrate concentrations by lipocortin isolated from human monocytes. However, at higher substrate concentrations, the inhibition disappeared, suggesting either that lipocortin sequestered the phospholipid substrate or that the putative inactive complex of enzyme and lipocortin dissociated in the presence of excess phospholipids. The hydrolysis of the neutral phospholipid phosphatidylethanolamine was stimulated by the presence of cardiolipin and phosphatidylglycerol. It is unlikely that this is caused merely by the negative charge of these phospholipids, since other negatively charged phospholipids did not show this effect. Using a phospholipid extract from mitochondria as substrate, the enzymatic activity as a function of the Ca2+ concentration was determined. Only one enzyme activity plateau was observed. The calculated KCa2+ value of 0.05 mM suggests that the mitochondrial phospholipase A2 could be regulated strictly by the modulation of the free Ca2+ concentration in vivo. The two activity plateaus observed previously upon variation of the Ca2+ concentration using phosphatidylethanolamine as substrate could be explained by a Ca2+-induced transition of the phospholipid structure.     

Differentiation of phospholipases A in mitochondria and lysosomes of rat liver

Highly purified mitochondria from rat liver contain a phospholipase A that catalyzes removal of 2-fatty acids, with a pH optimum above pH 8.0. Lysosomal preparations appeared to have two phospholipases A associated with them, one with a pH optimum at about pH 4.0, the second between pH 6.0 and 7.0. Mitochondrial phospholipase A hydrolyzed exogenous phospholipid as fast as or faster than endogenous phospholipid. The difference in specific radioactivity of (14)C-ethanolamine-labeled endogenous mitochondrial phospholipid before and after incubation indicates that a fraction of mitochondrial phosphatidyl ethanolamine is hydrolyzed more rapidly than the mitochondrial phospholipids as a whole. Acyl bond hydrolysis of exogenous and endogenous phospholipid by mitochondria was stimulated by free fatty acid, Ca(++), or in certain cases, monoacyl phospholipids or by treatments that disrupt the mitochondrial membrane. Of various fatty acids tested, lauric, myristic, oleic, and linoleic were most effective. ADP and ATP inhibited mitochondrial phospholipase, probably because they compete for Ca(++). Mg(++) also behaved as a competitive inhibitor; the effect was overcome by relatively little Ca(++).     

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.     

Effect of detergents,trypsin, and bivalent metal ions on interfacial activation and functioning of phospholipase D

The effects of detergents, trypsin, and bivalent metal ions on production of phosphatidic and lysophosphatidic acids by the action of phospholipase D (PLD) on lecithin and lysolecithin were studied. It was found that these reaction products and dodecyl sulfate ions activate PLD, whereas other anionic detergents are less effective. A protective effect of the functioning enzyme against its hydrolytic inactivation by trypsin was found. Bivalent metal ions can be arranged in the following sequence by their ability to activate PLD in the hydrolysis of lecithin and lysolecithin: Ca²⁺ > Sr²⁺ > Ba²⁺ > Mg²⁺. These results are considered in relation to a proposed mechanism of activation and functioning of PLD with the participation of clusters of phosphatidates and lysophosphatidates. Such Me²⁺-induced formation of rafts or microdomains from the products of hydrolysis of phospholipids can rationalize not only PLD activation and self-regulation, but also the action of this mechanism on other components and properties of biomembranes. PLD and other lipolytic enzymes can be classified as lateral vector enzymes.     

Relation of lipid structure of sarcotubular vesicles to Ca++ transport activity

The role of lipids of the sarcotubular membranes in their Ca(++) uptake and Mg-ATPase activities was investigated. Treatment of the membranes with phospholipase C inhibits both processes. Treatment with phospholipase A and phospholipase D, which results in massive hydrolysis of the sarcotubular phospholipids, does not inhibit either the Ca(++) uptake or the Mg-ATPase activities, nor does treatment with the polyene antibiotics affect these processes. Essential fatty acid deficiency alters sarcotubular membrane lipids; they contain much less stearic, linoleic, and arachidonic acids and much more oleic and eicosatrienoic acids than normally, but do not lose the ability to actively sequester Ca(++). It is concluded that neither nonpolar lipids nor the nonpolar regions of polar lipids are involved in Ca(++) sequestering and Mg-ATPase activities of the sarcotubular membranes. Of the polar components, the phosphoryl moiety of the phospholipids is required for both activities. However, the phosphoryl group appears to be required for the maintenance of the membranous structure necessary for Ca(++) sequestration rather than serving specifically in the active transport process. That treatment with phospholipase D, which results in the conversion of much of the sarcotubular phospholipid from a dipolar to an anionic structure, does not affect Ca(++) uptake activity is a most remarkable finding.     

Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state

Short-chain lecithin/long-chain phospholipid unilamellar vesicles (SLUVs), unlike pure long-chain lecithin vesicles, are excellent substrates for water-soluble phospholipases. Hemolysis assays show that greater than 99.5% of the short-chain lecithin is partitioned in the bilayer. In these binary component vesicles, the short-chain species is the preferred substrate, while the long-chain phospholipid can be treated as an inhibitor (phospholipase C) or poor substrate (phospholipase A2). For phospholipase C Bacillus cereus, apparent Km and Vmax values show that bilayer-solubilized diheptanoylphosphatidylcholine (diheptanoyl-PC) is nearly as good a substrate as pure micellar diheptanoyl-PC, although the extent of short-chain lecithin hydrolysis depends on the phase state of the long-chain lipid. For phospholipase A2 Naja naja naja, both Km and Vmax values show a greater range: in a gel-state matrix, diheptanoyl-PC is hydrolyzed with micellelike kinetic parameters; in a liquid-crystalline matrix, the short-chain lecithin becomes comparable to the long-chain component. Both enzymes also show an anomalous increase in specific activity toward diheptanoyl-PC around the phase transition temperature of the long-chain phospholipid. Since the short-chain lecithin does not exhibit a phase transition, this must reflect fluctuations in head-group area or vertical motions of the short-chain lecithin caused by surrounding long-chain lecithin molecules. These results are discussed in terms of a specific model for SLUV hydrolysis and a general explanation for the "interfacial activation" observed with water-soluble phospholipases.     

Binding of divalent and trivalent cations with crotoxin and with its phospholipase and its non-catalytic subunits: effects on enzymatic activity and on the interaction of phospholipase component with phospholipids

We have studied the interaction of divalent and trivalent with a potent phospholipase A(2) neurotoxin, crotoxin, from Crotalus durissus terrificus venom. The pharmacological action of crotoxin requires dissociation of its catalytic subunit (component B) and of its non-enzymatic chaperone subunit (component A), then the binding of the phospholipase subunit to target sites on cellular membranes and finally phospholipid hydrolysis. In this report, we show that the phospholipase A(2) activity of crotoxin and of component B required Ca2+ and that other divalent cations (Sr2+, Cd2+ and Ba2+) and trivalent lanthanide ions are inhibitors. The lowest phospholipase A(2) activity was observed in the presence of Ba2+, which proved to be a competitive inhibitor of Ca2+. The binding of divalent cations and trivalent lanthanide ions to crotoxin and to its subunits has been examined by equilibrium dialysis and by spectrofluorimetric methods. We found that crotoxin binds two divalent cations per mole with different affinities; the site presenting the highest affinity (K(d) in the mM range) in involved in the activation (or inhibition) of the phospholipase A(2) activity and must therefore be located on component B, the other site (K(d) higher than 10 mM) is probably localized on component A and does not play any role in the catalytic activity of crotoxin. We also observed that crotoxin component B binds to vesicular and micellar phospholipids, even in the absence of divalent cations. The affinity of this interaction either does not change or else increases by an order of magnitude in the presence of divalent cations.     

Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR

The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.     

Phospholipids of rat tissues after feeding pure phosphatidyl ethanolamine and lecithin

Pure phosphatidyl ethanolamine and lecithin from egg yolks were fed to rats in saline or in olive oil and the changes in individual phospholipids in the intestinal wall, liver, and plasma of the animals were studied. Ingestion of olive oil alone produced increased levels of all phospholipid fractions in each of the three tissues. Feeding phosphatidyl ethanolamine in saline resulted in slightly increased plasma phospholipids, but levels of liver total phospholipids were greatly reduced; when phosphatidyl ethanolamine was fed with olive oil, liver phospholipids were again reduced but this reduction was confined to the phosphatidyl ethanolamine and phosphatidic acid fractions. Feeding lecithin alone did not produce significant changes in levels of plasma or tissue phospholipids. The results suggest that liver phospholipid synthesis is depressed by feeding phosphatidyl ethanolamine; in the presence of olive oil, hepatic synthesis of phosphatidyl ethanolamine seems to be more selectively inhibited.     

The chemical basis for interfacial activation of monomeric phospholipases A2. Autocatalytic derivatization of the enzyme by acyl transfer from substrate

A basic monomeric phospholipase A2 from the venom of the American water moccasin, Agkistrodon piscivorus piscivorus, undergoes Ca2+-dependent, autocatalytic acylation during the course of hydrolysis of both model and natural phospholipid substrates. Acylation occurs at 2 lysine residues, Lys-7 and Lys-10, in the NH2-terminal alpha-helical segment of the enzyme, and when both positions are fully derivatized, the stable bisacylphospholipase A2 becomes a dimer in solution. The acylated enzyme is fully activated toward monomolecular layers of lecithins. Similar studies applied to the monomeric phospholipases A2 from porcine pancreas and from the venom of Agkistrodon contortrix contortrix also showed irreversible activation of the enzymes by substrate with the same kinetic consequences and formation of dimers. Acylation thus enables these enzymes to overcome the lag period observed under such conditions with native monomeric phospholipases, a phenomenon referred to as interfacial activation. Activation of the enzyme by acylation potentiates the phospholipase for interfacial recognition via formation of a dimeric enzyme. The naturally occurring phospholipase A2 dimer from Crotalus atrox venom displays no lag in the hydrolysis of lecithin monolayers nor does it undergo substrate level acylation. These facts support our proposal that dimerization concomitant with acylation is responsible for the large rate enhancements seen in the hydrolysis of aggregated phospholipids by monomeric phospholipases. Our findings demonstrate for the first time a chemical mechanism for interfacial activation of and interfacial recognition by phospholipases A2.     

Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

The role of intramembrane Ca2+ in the hydrolysis of the phospholipids of Escherichia coli by Ca2+-dependent phospholipases

Ca2+-dependent phospholipases A require Ca2+ concentrations in the millimolar range for optimal activity toward artificial substrates. Because Ca2+-dependent phospholipases A2 degrade the phospholipids of Escherichia coli, treated with the membrane-active antibiotic polymixin B equally well with and without added Ca2+ (Weiss, J., Beckerdite-Quagliata, S., and Elsbach, P. (1979) J. Biol. Chem. 254, 11010-11014), we have examined the possibility that intramembrane Ca2+ can provide the Ca2+ needed for phospholipase action. We studied the effect of Ca2+ depletion on the hydrolysis of the phospholipids of polymixin B-killed E. coli by 1) added pig pancreas phospholipase A2 in E. coli S17 (a phospholipase A-lacking mutant) and 2) endogenous Ca2+-dependent phospholipase A1 in the parent strain E. coli S15. Transfer of E. coli from nutrient broth (Ca2+ concentration approximately 3 X 10(-5) M) to Ca2+-depleted medium (Ca2+ concentration less than 10(-6)M) reduced polymixin B-induced hydrolysis by 50-75%, in parallel with a reduction of bacterial Ca2+ from 19.6 +/- 2.8 to 3.9 +/- 0.6 nmol (mean +/- standard error) per 3 X 10(10) bacteria. The bacterial Ca2+ content was repleted and the sensitivity of the bacterial phospholipids to hydrolysis by both exogenous phospholipase A2 (E. coli S17) and endogenous phospholipase A (E. coli S15) was restored by adding Ca2+ back to the suspensions. Complete restoration occurred at low Ca2+ levels in the reaction mixture (3 X 10(-5) - 10(-4) M) and required time, suggesting that hydrolysis was restored because bacterial Ca2+ stores were gradually replenished and not because extracellular Ca2+ concentrations were raised to levels that were still at least 10X lower than needed for optimal phospholipase A activity. This conclusion is supported by the finding that Ca2+ depletion or addition caused respectively decreased and increased release of lipopolysaccharides by EGTA (ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid), suggesting that the bacterial Ca2+ pool bound to lipopolysaccharides in the outer membrane shrinks or expands depending on extracellular Ca2+ levels. Thus, the cationic membrane-disruptive polymixin B, thought to compete with Mg2+ and Ca2+ for the same anionic sites on lipopolysaccharides, may liberate the Ca2+ near where the phospholipids are exposed to phospholipase.     

The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (cestoda)

Frayha G. J., Bahr G. M. and Haddad R. 1980. The lipids and phospholipids of hydatid protoscolices of Echinococcus granulosus (Cestoda). International Journal for Parasitology10: 213–216. The protoscolices of Echinococcus granulosus were isolated from hydatid cysts. The phospho- and neutral lipids of the protoscolices were determined. The seven major classes of lipids namely, phospholipids, fatty acids, mono-, di- and triglycerides, cholesterol and cholesterol esters were identified and quantitatively determined. The phospholipids isolated were cephalin, lecithin, lysolecithin, phosphatidyl inositol, phosphatidyl serine and sphingomyelin. Lecithin and cephalin were the most abundant. The signiflcance of this finding is discussed.     

The lipid requirement of the (Ca2+ + Mg2+)-ATPase in the human erythrocyte membrane, as studied by various highly purified phospholipases.

1. When complete hydrolysis of glycerophosphlipids and sphingomyelin in the outer membrane leaflet is brought about by treatment of intact red blood cells with phospholipase A2 and sphingomyelinase C, the (Ca2+ + Mg2+)-ATPase activity is not affected. 2. Complete hydrolysis of sphingomyelin, by treatment of leaky ghosts with spingomyelinase C, does not lead to an inactivation of the (Ca2+ + Mg2+)-ATPase. 3. Treatment of ghosts with phospholipase A2 (from either procine pancreas of Naja naja venom), under conditions causing an essentially complete hydrolysis of the total glycerophospholipid fraction of the membrane, results in inactivation of the (Ca2+ + Mg2+)-ATPase by some 80--85%. The residual activity is lost when the produced lyso-compounds (and fatty acids) are removed by subsequent treatment of the ghosts with bovine serum albumin. 4. The degree of inactivation of the (Ca2+ + Mg2+)-ATPase, caused by treatment of ghosts with phospholipase C, is directly proportional to the percentage by which the glycerophospholipid fraction in the inner membrane layer is degraded. 5. After essentially complete inactivation of the (Ca2+ + Mg2+)-ATPase by treatment of ghosts with phospholipase C from Bacillus cereus, the enzyme is reactivated by the addition of any of the glycerophospholipids, phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine or lysophosphatidylcholine, but not by addition of sphingomyeline, free fatty acids or the detergent Triton X-100. 6. It is concluded that only the glycerophospholipids in the human erythrocyte membrane are involved in the maintenance of the (Ca2+ + Mg2+)-ATPase activity, and in particular that fraction of these phospholipids located in the inner half of the membrane.     

Characterization of an 11,000-dalton beta-bungarotoxin: binding and enzyme activity on rat brain synaptosomal membranes

The binding and phospholipase A2 activity of an 11,000-dalton beta-bungarotoxin, isolated from Bungarus multicincutus venom, have been characterized using rat brain subcellular fractions as substrates. 125I-labeled beta-bungarotoxin binds rapidly (k = 0.14 min-1 and 0.11 min-1), saturably (Vmax = 130.1 +/- 5.0 fmoles/mg and 128.2 +/- 7.1) fmoles/mg), and with high affinity (apparent Kd = 0.8 +/- 0.1 nM and 0.7 +/- 0.1 nM) to rat brain mitochondria and synaptosomal membranes, respectively, but not to myelin. The binding to synaptosomal membranes is inhibited by divalent cations and by pretreatment with trypsin. The binding results suggest that the toxin binds to specific protein receptor sites on presynpatic membranes. The 11,000-dalton toxin rapidly hydrolyzes synaptosomal membrane phospholipids to lysophosphatides and manifests relative substrate specificity in the order phosphatidyl ethanolamine greater than phosphatidyl choline greater than phosphatidyl serine. These results indicate that the 11,000-dalton beta-bungarotoxin is a phospholipase A2 and can use presynaptic membrane phospholipids as substrates. The binding, phospholipase activity and other biological properties of the 11,000-dalton toxin are contrasted with those of the beta-bungarotoxin found in highest concentration in the venom (the 22,000-dalton beta-bungarotoxin), and the two toxins are shown to have qualitatively similar properties. Finally the results are shown to support the hypothesis that beta-bungarotoxins act in a two-step fashion to inhibit transmitter release: first, by binding to a protein receptor site on the presynatic membrane associated with Ca2+ entry, and second, by perturbing through enzymatic hydrolyses the phospholipid matrix of the membrane and thereby causing an increase in passive Ca2+ permeability.     

Promotion of conidia aggregation in Aspergillus niger by cyclic AMP and 5'-GMP

Cholesterol and phospholipids remain tightly associated with the ferroxidase-II protein from human serum following extensive purification. Purified ferroxidase-II preparations show a consistent ratio of protein, phospholipid, and cholesterol. Thin-layer chromatographic analyses indicate that phosphatidyl choline accounts for 70% of the bound phospholipid. Treatment of purified ferroxidase-II with phospholipase C or A results in a loss of ferroxidase activity which parallels the hydrolysis of phospholipid. A lipid-depleted form of ferroxidase-II can be prepared by gel-filtration following treatment with phospholipase C. However, hydrolysis, not removal, of the lipid is sufficient for the loss of ferroxidase activity. These studies indicate that the bound lipid components are essential to the maintainence of the catalytic activity of ferroxidase-II.