The action of human and rabbit serum phospholipase A2 on Escherichia coli phospholipids

We have compared the properties of phospholipase A (E.C. 3.1.1.4) activity in whole human and rabbit serum toward the phospholipids of Escherichia coli. Using as substrate E. coli labeled during growth with either [1-(14)C]-palmitic acid or [1-(14)C]oleic acid, and then autoclaved to inactivate E. coli phospholipases and to render the labeled phospholipids accessible to exogenous phospholipases, we show that the deacylating activity in both human and rabbit serum is almost exclusively of the A(2) type. Rabbit serum is at least 20-fold more active than human serum. Activity in both sera is maximal at physiological Ca(2+) concentrations (2 mM) and is abolished by ethylenediaminetetraacetic acid. To examine hydrolysis of intact (unautoclaved) E. coli treated with 25% serum, use was made of a phospholipase A-deficient E. coli strain (E. coli S17), thereby eliminating the possible contribution of bacterial phospholipases to degradation. Human and rabbit serum are about equally bactericidal toward E. coli and cause comparable structural damage. However, only rabbit serum produces substantial hydrolysis of the phospholipids of intact E. coli S17. Heated (56 degrees C, 30 min) rabbit serum is non-bactericidal and retains phospholipase A(2) activity toward autoclaved, but not intact E. coli. The ability of heated serum to degrade phospholipids of intact E. coli S17 is restored, however, by adding 25% normal human serum, which is bactericidal. In this combination, doses of heated rabbit serum containing as much phospholipase A(2) activity (toward autoclaved E. coli) as is present in 25% unheated rabbit serum, produce roughly the same extent of hydrolysis of intact E. coli as does normal rabbit serum alone. Low doses with a phospholipase A(2) activity comparable to that of normal human serum elicit little or no hydrolysis. These findings indicate that hydrolysis of the phospholipids of intact E. coli S17 by serum occurs when: 1) the serum is bactericidal, and 2) when sufficient phospholipase A(2) is present. The difference in phospholipid hydrolysis that accompanies killing of E. coli by human or rabbit serum appears to reflect, therefore, the different amounts of phospholipase A(2) activity in the two sera. Phospholipid degradation is not required for the bactericidal action of serum. Bacterial phospholipid breakdown may be important, however, in the overall destruction and digestion of invading bacteria by the host.-Kaplan-Harris, L., J. Weiss, C. Mooney, S. Beckerdite-Quagliata, and P. Elsbach. The action of human and rabbit serum phospholipase A(2) on Escherichia coli phospholipids.     

Relation between binding and the action of phospholipases A2 on Escherichia coli exposed to the bactericidal/permeability-increasing protein of neutrophils

Exposure of Escherichia coli to the bactericidal/permeability-increasing protein (BPI) of neutrophils renders the bacterial phospholipids susceptible to hydrolysis by only a few of numerous phospholipases A2 tested. To explore further the determinants of hydrolysis we measured the binding of 125I-labeled phospholipase A2 to E. coli in the presence and absence of BPI. Phospholipases A2 from Aqkistrodon piscivorus piscivorus venom and pig pancreas neither degraded nor bound to BPI-treated E. coli. In contrast, the phospholipases A2 from Aqkistrodon halys blomhoffii and Aqkistrodon halys palas venoms actively hydrolyzed the phospholipids of BPI-treated E. coli: they also bound to E. coli in the presence but not in the absence of BPI. Carbamylation of lysines of the A.h. blomhoffii phospholipase A2 progressively reduced binding in parallel with reduced phospholipid hydrolysis. Both binding and hydrolysis increased with increasing BPI dose. However, maximal binding occurred at 25% of the BPI dose that produced optimal hydrolysis. Thus, binding may be necessary but is not sufficient for maximal BPI-mediated phospholipid hydrolysis. Comparison of the NH2-terminal amino sequences of the active and inactive phospholipase A2 suggests that this portion of the phospholipase A2 molecule plays a role in BPI-independent binding and hydrolysis.     

Quantitative determination of Ca2+-dependent Mg2+-ATPase from sarcoplasmic reticulum in muscle biopsies.

The possibility of quantifying the total concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum was investigated by measurement of the Ca2+-dependent steady-state phosphorylation from [gamma-32P]ATP and the Ca2+-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity in crude muscle homogenates. The Ca2+-dependent phosphorylation at 0 degree C (mean +/- S.E.) was 40.0 +/- 2.5 (n = 6) and 6.2 +/- 0.7 (n = 4) nmol/g wet wt. in rat extensor digitorum longus (EDL) and soleus muscle, respectively (P less than 0.001). The Ca2+-dependent 3-O-MFPase activity at 37 degrees C was 1424 +/- 238 (n = 6) and 335 +/- 56 (n = 4) nmol/min per g wet wt. in rat EDL and soleus muscle, respectively (P less than 0.01). The molecular activity calculated from these measurements amounted to 35 +/- 5 min-1 (n = 6) and 55 +/- 10 min-1 (n = 4) for EDL and soleus muscle respectively. These values were not different from the molecular activity calculated for purified Ca2+-ATPase (36 min-1). The Ca2+-dependent 32P incorporation in soleus muscle decreased in the order mice greater than rats greater than guinea pigs. In EDL muscles from hypothyroid rats at a 30% reduction of the Ca2+-dependent phosphorylation was observed. The Ca2+-dependent phosphorylation in vastus lateralis muscle from three human subjects amounted to 4.5 +/- 0.8 nmol/g wet wt. It is concluded that measurement of the Ca2+-dependent phosphorylation allows rapid and reproducible quantification of the concentration of Ca2+-dependent Mg2+-ATPase of sarcoplasmic reticulum. Since only 20-60 mg of tissue is required for the measurements, the method can also be used for biopsies obtained in clinical studies.     

Rabbit platelet calcium ATPase differs from the human erythrocyte (Ca2+ + Mg2+)-ATPase in its response to three purified phospholipases A2, exogenous phospholipids and calmodulin

Human erythrocyte (Ca2+ + Mg2+)-ATPase and calcium ATPase of rabbit platelets were compared by their responses to a variety of treatments. These included three purified phospholipases A2 (acidic, neutral and basic) from Agkistrodon halys blomhoffii, as well as several phospholipids and lysophospholipids. The erythrocyte enzyme was stimulated 2-3-fold by all three phospholipases with maximal stimulation occurring at different concentrations of the three enzymes. The basic phospholipase was the most potent, followed by the neutral and acidic enzymes in that order. The calcium ATPase activity of the platelet was also stimulated by phospholipase treatment, but only by 10-20%. The stimulatory activity was attributable to hydrolysis of a very small portion of the total membrane phospholipid. Inactivation of the phospholipases by heating or chemical modification with p-bromophenacyl bromide abolished their ability to stimulate. Addition of polyphosphoinositides stimulated both ATPases. However, another acidic phospholipid, lysophosphatidic acid, stimulated only the erythrocyte enzyme and failed to affect the platelet calcium ATPase. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) had no effect on either enzyme, while the platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine), its lyso compound and lysoPC inhibited both ATPases. Calmodulin stimulated the erythrocyte enzyme, but did not affect the platelet calcium ATPase. These results demonstrate that the protein-lipid interactions operative in the erythrocyte and platelet calcium ATPases are quite different.     

No direct correlation between Ca2+ mobilization and dissociation of Gi during platelet phospholipase A2 activation

Stimulation of human platelets with thrombin is accompanied by activation of both phospholipases C and A2. These have been considered to be sequential events, with phospholipase A2 activation resulting from the prior hydrolysis of inositol phospholipids and mobilization of intracellular Ca2+ stores. However, our and other laboratories have recently questioned this proposal, and we now present further evidence that these enzymes may be activated by separate mechanisms during thrombin stimulation. Alpha-thrombin induced the rapid hydrolysis of inositol phospholipids, and formation of inositol trisphosphate and phosphatidic acid. This was paralleled by mobilization of Ca2+ from internal stores. These responses were blocked by about 50% by prostacyclin. In contrast, the liberation of arachidonic acid induced by alpha-thrombin was totally inhibited by prostacyclin. The less-effective agonists, platelet activating factor (PAF) and gamma-thrombin also both stimulated phospholipase C, but whereas PAF evoked a rapid and transient response, that of gamma-thrombin was delayed and more sustained. The abilities of these agonists to induce the release of Ca2+ stores closely paralleled phospholipase C activation. However, the maximal intracellular Ca2+ concentrations achieved by these two agents were the same. Despite this, gamma-thrombin and not PAF, was able to release a small amount of arachidonic acid. When alpha-thrombin stimulation of platelets was preceded by epinephrine, there was a potentiation of phospholipase C activation, Ca2+ mobilization and aggregation. The same was true for gamma-thrombin and PAF. However, unlike alpha-thrombin, the gamma-thrombin-stimulated arachidonic acid release was not potentiated by epinephrine, but rather somewhat reduced. These results suggested that phospholipase C and phospholipase A2 were separable events in activated platelets. The mechanism by which alpha-thrombin stimulated phospholipase A2 did not appear to be through dissociation of the inhibitory GTP-binding protein, Gi, since gamma-thrombin decreased the pertussis toxin-induced ADP-ribosylation of the 41 kDa protein as much as did alpha-thrombin, but was a much less effective agent than alpha-thrombin at inducing arachidonic acid liberation.     

Stimulation of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by phospholipase D

Treatment of canine cardiac sarcolemmal vesicles with phospholipase D resulted in a large stimulation (up to 400%) of Na+-Ca2+ exchange activity. The phospholipase D treatment decreased the apparent Km (Ca2+) for the initial rate of Nai+-dependent Ca2+ uptake from 18.2 +/- 2.6 to 6.3 +/- 0.3 microM. The Vmax increased from 18.0 +/- 3.6 to 31.5 +/- 3.6 nmol of Ca2+/mg of protein/s. The effect was specific for Na+-Ca2+ exchange; other sarcolemmal transport enzymes ((Na+, K+)-ATPase; ATP-dependent Ca2+ transport) are inhibited by incubation with phospholipase D. Phospholipase D had little effect on the passive Ca2+ permeability of the sarcolemmal vesicles. After treatment with 0.4 unit/ml of phospholipase D (20 min, 37 degrees C), the sarcolemmal content of phosphatidic acid rose from 0.9 +/- 0.2 to 8.9 +/- 0.4%; simultaneously, Na+-Ca2+ exchange activity increased 327 +/- 87%. It is probable that the elevated phosphatidic acid level is responsible for the enhanced Na+-Ca2+ exchange activity. In a previous study (Philipson, K. D., Frank, J. S., and Nishimoto, A. Y. (1983) J. Biol. Chem. 258, 5905-5910), we hypothesized that negatively charged phospholipids were important in Na+-Ca2+ exchange, and the present results are consistent with this hypothesis. Stimulation of Na+-Ca2+ exchange by phosphatidic acid may be important in explaining the Ca2+ influx which accompanies the phosphatidylinositol turnover response which occurs in a wide variety of tissues.     

High affinity Ca2+ -Mg2+ ATPase in the distal tubule of the mouse kidney

The purpose of this study was to investigate whether Ca2+ -Mg2+ ATPase in the distal tubule (where calcium transport is active, against a gradient, and hormone dependent) presents some characteristics different from those observed in the proximal tubule, and whether these characteristics are likely to shed light on the respective roles of this enzyme at the two sites of the nephron. The Ca2+ - and Mg2+-dependent ATP hydrolysis was measured in microdissected segments of the distal nephron, the kinetic parameters were determined, and the influence of magnesium upon the sensitivity to calcium was examined. Results were compared with those obtained in the proximal tubule, and in purified membranes as reported by others. In the distal tubule, low concentrations of Mg2+ (less than 10(-7) M) did not influence ATP hydrolysis. At concentrations above 10(-7) M, Mg2+ increased ATP hydrolysis according to Michaelis kinetics (apparent Km = 11.3 +/- 2.4 microM, Vmax = 219 +/- 26 pmol.mm-1.20 min-1). The addition of 1 microM Ca2+ decreased the apparent Km for Mg2+ and the Vmax for Mg2+. Similar results were obtained in the proximal tubule. At low Mg2+ concentrations, Ca2+ also stimulated ATP hydrolysis according to Michaelis kinetics with an apparent Km value for Ca2+ of 0.18 +/- 0.06 and 0.10 +/- 0.03 microM Ca2+ (ns) and a Vmax of 101 +/- 12 and 89 +/- 9 pmol.mm-1.20 min-1 (ns) in the distal and proximal tubules, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells

The pathways for degradation of phosphatidylinositol (PI) were investigated in sonicated suspensions prepared from confluent cultures of bovine pulmonary artery endothelial cells. The time courses of formation of 3H-labeled and 14C-labeled metabolites of phosphatidyl-[3H]inositol ([3H]Ins-PI) and 1-stearoyl-2-[14C] arachidonoyl-PI were determined at 37 degrees C and pH 7.5 in the presence of 2 mM EDTA with or without a 2 mM excess of Ca2+. The rates of formation of lysophosphatidyl-[3H]inositol ([3H]Ins-lyso-PI) and 1-lyso-2-[14C] arachidonoyl-PI were similar in the presence and absence of Ca2+, and the absolute amounts of the two radiolabeled lyso-PI products formed were nearly identical. This indicated that lyso-PI was formed by phospholipase A1, and phospholipase A2 was not measurable. In the presence of EDTA, [14C]arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI paralleled release of glycerophospho-[3H]inositol ([3H]GPI) from [3H]Ins-PI. Formation of [3H]GPI was inhibited by treatment with the specific sulfhydryl reagent, 2,2'-dithiodipyridine, and this was accompanied by an increase in [3H]Ins-lyso-PI. In the presence of Ca2+, [14C] arachidonic acid release from 1-stearoyl-2-[14C]arachidonoyl-PI was increased 2-fold and was associated with Ca2+-dependent phospholipase C activity. Under these conditions, [3H]inositol monophosphate production exceeded formation of [14C]arachidonic acid-labeled phospholipase C products, diacylglycerol plus monoacylglycerol, by an amount that was equal to the amount of [14C]arachidonic acid formed in excess of [3H]GPI. Low concentrations of phenylmethanesulfonyl fluoride (15-125 microM) inhibited Ca2+-dependent [14C]arachidonic acid release, and the decrease in [14C] arachidonic acid formed was matched by an equivalent increase in 14C label in diacylglycerol plus monoacyclglycerol. These data supported the existence of two pathways for arachidonic acid release from PI in endothelial cells; a phospholipase A1-lysophospholipase pathway that was Ca2+-independent and a phospholipase C-diacylglycerol lipase pathway that was Ca2+-dependent. The mean percentage of arachidonic acid released from PI via the phospholipase C-diacylglycerol lipase pathway in the presence of Ca2+ was 65 +/- 8%. The mean percentage of nonpolar phospholipase C products of PI metabolized via the diacylglycerol lipase pathway to free arachidonic acid was 28 +/- 3%.     

Insights into metal ion binding in phospholipases A2: ultra high-resolution crystal structures of an acidic phospholipase A2 in the Ca2+ free and bound states

The electrophile Ca(2+) is an essential multifunctional co-factor in the phospholipase A(2) mediated hydrolysis of phospholipids. Crystal structures of an acidic phospholipase A(2) from the venom of Bothrops jararacussu have been determined both in the Ca(2+) free and bound states at 0.97 and 1.60 A resolutions, respectively. In the Ca(2+) bound state, the Ca(2+) ion is penta-coordinated by a distorted pyramidal cage of oxygen and nitrogen atoms that is significantly different to that observed in structures of other Group I/II phospholipases A(2). In the absence of Ca(2+), a water molecule occupies the position of the Ca(2+) ion and the side chain of Asp49 and the calcium-binding loop adopts a different conformation.     

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 graulocytes 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 micrograms of granulocyte fraction causes loss of viability of from 90 to 99% of 1 X 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.     

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.     

Amino acid sequence of a basic Agkistrodon halys blomhoffii phospholipase A2. Possible role of NH2-terminal lysines in action on phospholipids of Escherichia coli

A basic (pI = 10.2) phospholipase A2 of the venom of the snake Agkistrodon halys blomhoffii is one of a few phospholipases A2 capable of hydrolyzing the phospholipids of Escherichia coli killed by a bactericidal protein purified from human or rabbit neutrophil granules. We have shown that modification of as many as 4 mol of lysine per mole of the phospholipase A2, either by carbamylation or by reductive methylation [Forst, S., Weiss, J., & Elsbach, P. (1982) J. Biol. Chem. 257, 14055-14057], had no effect on catalytic activity toward extracted E. coli phospholipids or the phospholipids of autoclaved E. coli. In contrast, modification of 1 mol of lysine per mole of enzyme substantially reduced activity toward the phospholipids of E. coli killed by the neutrophil protein. To explore further the role of lysines in the function of this phospholipase A2, we determined the amino acid sequence of the enzyme and the incorporation of [14C]cyanate into individual lysines when, on average, 1 lysine per molecule of enzyme had been carbamylated. After incorporation of approximately 1 mol of [14C]cyanate per mole of protein, the phospholipase A2 was reduced, alkylated, and exhaustively carbamylated with unlabeled cyanate. The amino acid sequence was determined of the NH2-terminal 33 amino acids of the holoprotein and of peptides isolated after digestion with trypsin and Staphylococcus aureus V-8 protease. The protein contains 122 amino acid residues, 17 of which are lysines. The NH2-terminal region is unique among more than 30 phospholipases A2 previously sequenced because of its high content of basic residues (His-1, Arg-6, and Lys-7, -10, -11, and -15).(ABSTRACT TRUNCATED AT 250 WORDS)     

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.     

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of calcium

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of Ca2+ was studied. At pH 6.0, 10 degrees C and in the absence of K+, the enzyme displays a very low velocity of ATP hydrolysis. Addition of up to 15% dimethyl sulfoxide increased this velocity severalfold (from 5-18 nmol of Pi X mg of protein-1 X h-1) and then decreased at higher solvent concentrations. Dimethyl sulfoxide increased both enzyme phosphorylation from ATP and the affinity for this substrate. Maximal levels of 1.0-1.2 nmol of EP X mg of protein-1 and apparent KM for ATP of 5 X 10(-6) M were obtained at a concentration of 30% dimethyl sulfoxide. The same preparation under optimal conditions (pH 7.5, 10 microM CaCl2, 100 mM KCl and no dimethyl sulfoxide at 37 degrees C) displays a velocity of ATP hydrolysis between 8 and 12 X 10(5) nmol of Pi X mg of protein-1 X h-1 while the phosphoenzyme levels varied between 3.5 and 4.0 nmol of EP X mg of protein-1. Enzyme phosphorylation from ATP in the absence of Ca2+ always preceded Pi liberation into the assay media. Two different phosphoenzyme species were formed which were kinetically distinguished by their decomposition rates. The observed steady-state velocity of ATP hydrolysis could be accounted for either by the decay of the fast component or by the simultaneous decomposition of both phosphoenzyme species. The hydrolysis of the phosphoenzyme formed in the absence of Ca2+ was KCl-stimulated and ADP-independent. The rate constant of breakdown was equal to that observed for the phosphoenzyme formed in the presence of Ca2+. It is suggested that the rapidly decaying phosphoenzyme (and possibly both rapidly and slowly decaying species) are intermediates in the reaction cycle of Mg2+-dependent ATP hydrolysis of sarcoplasmic reticulum Ca2+-ATPase and may represent a bypass of Ca2+ activation by dimethyl sulfoxide.     

Effects of antimalarial drugs on phospholipase A and lysophospholipase activities in plasma membrane, mitochondrial, microsomal and cytosolic subcellular fractions of rat liver

Activities of membrane-associated phospholipases A1 and A2, and membrane-associated as well as soluble lysophospholipases were measured in different subcellular fractions of rat liver, using suspensions of stereospecifically labelled radioactive phospholipids as substrates. Plasma membranes and endoplasmic reticulum were shown to contain phospholipase A1 and lysophospholipase activities, both of which could be stimulated by Ca2+, mitochondria Ca2+-dependent phospholipase A2 and cytosol Ca2+-independent lysophospholipase activities. Each of these lipolytic enzymes could be inhibited by antimalarial drugs (chloroquine, mepacrine, primaquine) at concentrations above 1 x 10(-4) M. Inhibition of the alkaline cytosolic lysophospholipase by these drugs was noncompetitive with respect to the substrate, and the inhibitory potency increased, when the pH was raised.     

The relationship between high-affinity noncatalytic binding of snake venom phospholipases A2 to brain synaptic plasma membranes and their central lethal potencies

The basic phospholipase A2 from Naja nigricollis (African spitting cobra) snake venom is enzymatically less active but more toxic than the acidic phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom, following injection into the right lateral ventricle of the brain of rats. When radiolabeled with 125I, these phospholipases A2 retained enzymatic activities and lethal potencies. Both enzymes bound with high affinity and specificity to brain synaptic plasma membrane preparations in vitro even in the absence of calcium, suggesting a non-catalytic binding. The acidic enzyme, in a calcium-free medium, had two binding components with Kd values of 1 X 10(-10) and 2.75 X 10(-8) M and Bmax values of 6 X 10(-13) and 3.4 X 10(-11) mol/mg, respectively. Multiple specific and nonspecific binding components were observed for each phospholipase A2; saturability for all of the binding sites was conclusively demonstrated only for the N. naja atra phospholipase A2 in a calcium-free medium (Bmax = 3.4 X 10(-11) mol/mg). The levels of specific and total binding were 150 pmol/mg and 450 pmol/mg, respectively, for the comparatively toxic enzyme and 15 pmol/mg and 35 pmol/mg, respectively, for the comparatively nontoxic enzyme at a concentration of 2.5 X 10(-8) M. These levels of binding (both total and specific) were directly correlated with the intraventricular lethal potencies of the phospholipases A2 (0.5 and 5.0 micrograms/rat for the N. nigricollis and N. naja atra phospholipases A2, respectively), suggesting a possible relationship between binding and lethal potency. Carbamylation of lysines reduced the levels of binding and the lethal potencies of both enzymes to a greater extent than their enzymatic activities. Pretreatment with high temperature, proteinases, phospholipases A2 or C suggested that radiolabeled phospholipase A2 binds to phospholipids rather than proteins. However, only the N. naja atra phospholipase A2 manifested a strict dependence on a divalent cation (Ca2+ or Sr2+) for most of its binding. The N. nigricollis enzyme demonstrated a much lower rate of dissociation from synaptic plasma membranes than did N. naja atra phospholipase A2, suggesting that hydrophobic interactions are more important in the binding of the more toxic enzyme as compared to the less toxic enzyme. It is proposed that differences in the extent of high-affinity noncatalytic binding to membrane phospholipids may be at least partly responsible for the marked difference in central toxicities of these two phospholipases A2.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

1-14C]oleate-labeled autoclaved yeast: a membranous substrate for measuring phospholipase A2 activity in vitro

Radiolabeled, autoclaved yeast were tested as a substrate for mammalian phospholipase A2 activity because the only other membranous substrate used for this purpose, autoclaved Escherichia coli, totally lacks a major mammalian phospholipid, phosphatidylcholine. Candida albicans were grown in the presence of [1-14C]oleate and then autoclaved. Sixty three percent of the incorporated label was in yeast phospholipid, and more than 95% of that was in the 2-acyl position. The distribution of label in the yeast phospholipids (phosphatidylcholine and -ethanolamine, -serine + -inositol, and phosphatidic acid corresponded closely to the chemical distribution of phosphorus in those phospholipids. Snake venom (Naja naja) and human synovial fluid phospholipase A2 hydrolyzed yeast phospholipid exclusively to release 14C-labeled fatty acid. When 50-60% of the yeast phospholipid was hydrolyzed, the radioactive fatty acids as determined by gas-liquid chromatographic analysis were predominantly oleate (45%) and linoleate (greater than 54%). Hydrolysis of yeast phospholipid by both enzymes was near-linear with protein and time under conditions of optimal pH (neutral-alkaline) and Ca2- (1-5 mM) previously reported for optimal hydrolysis of autoclaved E. coli phospholipid. N. naja phospholipase A2 showed less preference for phosphatidylethanolamine than -choline as liposomes or yeast phospholipid as compared to human synovial fluid phospholipase A2 which clearly preferred phosphatidylethanolamine to -choline as a liposome or yeast phospholipid. These results illustrate that radiolabeled phospholipids of autoclaved yeast, enriched in phosphatidylcholine, are readily hydrolyzed by snake venom and human nonpancreatic phospholipases A2 and may, therefore, be useful in the measurement of in vitro enzymatic activity.