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PubMedDate not supplied

Interaction of epidermal growth factor with vasoactive hormones in the regulation of phospholipase A2.

The interaction of growth factors with their receptors initiates a series of intracellular events that are of critical importance in the control of normal cell proliferation. In this regard considerable attention has focused on the coupling of phospholipase C-gamma to growth factor receptor tyrosine kinases. In contrast, the interaction of growth factors with phospholipase A2 has received less attention, most likely because the arachidonic acid release response has been considered to be an accompaniment of phospholipase C activation. Work from our laboratory using a well defined model system demonstrated a distinct coupling relationship of epidermal growth factor to phospholipase A2. This review focuses on the interaction of the epidermal growth factor receptor with phospholipases involved in both mitogenic and non-mitogenic responses and discusses their possible relation with vasoactive hormones.

Angiotensin II↗
PubMed1980

Phospholipase A2 inhibitors and their possible clinical use in the treatment of acute pancreatitis.

A laboratory method was established for measurement of phospholipase A2 activity in buffer and serum. A series of different phospholipase A2 inhibitors was tested. The most effective inhibitors were Ca2+ chelating compounds like EDTA, DTPA, EGTA, and phytic acid. The calcium salt of EDTA also has some inhibitory effect. Serum phospholipase A2 activity in normal healthy control patients was measured. The activity in 27 patients with acute pancreatitis was tested. The activity was abnormally high in five patients. This activity was in vitro inhibited by EDTA and partly by CaNa2EDTA. The clinical picture of these patients did not differ from that of phospholipase-A2-negative patients. Six patients with acute pancreatitis were treated by intravenous infusion of CaNA2EDTA. Two of them had haemorrhagic pancreatitis and two were suspected of having early haemorrhagic pancreatitis. During the CaNa2EDTA infusion serum amylase and phospholipase A2 activities decreased. All patients recovered. No harmful side effects were noticed.

Acute Disease↗
PubMed1985

Studies of the phospholipase A2 activity of rat ileal mucosa.

A rapid and simple procedure has been used to determine phospholipase A2 activity (EC 3.1.1.4) in rat ileal mucosa. We used 14C-oleate-labeled Escherichia coli as substrate for the phospholipase activity and a 0.45-micron Millipore filter to separate the product of hydrolysis--the 14C-oleic acid--from the unhydrolyzed substrate. The phospholipase A2 activity was optimal at pH 9.8 and at 2 mM Ca2+, but another peak of activity appeared at pH 7.2. In addition, cell fractionation revealed yet another phospholipase A2 activity at pH 5.0 in the absence of Ca2+. These findings suggest the presence of more than one phospholipase A2 in the ileal mucosa and points to the possible use of a simple procedure for studying their distribution and properties.

Animals↗
PubMed2000

Evidence for phospholipases from Trypanosoma cruzi active on phosphatidylinositol and inositolphosphoceramide.

The lipid moiety in the glycosylphosphatidylinositol anchors of glycoproteins of Trypanosoma cruzi consists of an alkylacylglycerol, a lysoalkylglycerol or a ceramide. Previously, we showed that the inositolphosphoceramides (IPCs) are the major components in the precursor inositolphospholipids of epimastigote and trypomastigote forms. Using (3)H-labelled subfractions of IPC, phosphatidylinositol (PI) and glycoinositolphospholipids (GIPLs) as substrates with a cell-free system, we now demonstrate the association of at least five enzyme activities with the trypanosomal membranous particulate material. These include: phospholipase A(1) and phospholipase A(2), enzymes that release free fatty acid from the PI and GIPLs; an acyltransferase responsible for the acylation of the generated monoacyl or monoalkylglycerolipids with endogenous unlabelled fatty acid; two activities of phospholipase C, one releasing ceramide from IPC and the other alkylacylglycerol, alkylglycerol or diacylglycerol from PI. The neutral lipids were also generated on incubation of the GIPLs. The phospholipase C activities were inhibited by p-chloromercuriphenylsulphonic acid, as reported for other PI phospholipases C. An IPC-fatty-acid hydrolase, releasing fatty acid from the labelled IPC, was also observed. The enzyme activities reported in the present study may be acting in remodelling reactions leading to the anchor of the mature glycoproteins of T. cruzi.

Animals↗
PubMed1992

Inhibition of human phospholipase C by aurothiomalate and (triethylphosphine) gold complexes.

The effect of several gold complexes on the activity of phospholipase C from human blood platelets was studied in vitro. Aurothiomalate and auranofin--2 agents used for the chrysotherapy of rheumatoid arthritis--, gold chloride, (triethylphosphine)gold chloride, and 5 newly synthesized (triethylphosphine)gold complexes dose-dependently inhibited the enzyme with IC50 values between 0.8 mumol/l ((triethylphosphine)gold chloride) and over 100 mumol/l (auranofin). None of the compounds affected phospholipase A2 from human polymorphonuclear leukocytes at concentrations up to 100 mumol/l. Inhibition of phospholipase C by (triethylphosphine)gold chloride, aurothiomalate, and compound 3 was not significantly different at substrate concentrations of 20 and 100 mumol/l phosphatidylinositol, suggesting that these gold complexes do not inhibit phospholipase C by competing with the substrate. After confirming the Ca2+ dependence of the human platelet phospholipase C by demonstrating inhibition by the Ca2+ chelators EDTA and EGTA--but no inhibition by the Zn2+ chelator 1,10-phenanthroline--the inhibition of the enzyme by (triethylphosphine)gold chloride, aurothiomalate, and compound 3 was studied at 3 different concentrations of Ca2+ in the range of 0.2 to 2 mmol/l. Inhibition by (triethylphosphine)gold chloride was not affected by changes of Ca2+, whereas inhibition by aurothiomalate and compound 3 was markedly suppressed by increasing the Ca2+ concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Antirheumatic Agents↗
PubMed1990

Release of dialkylglycerol from purple membrane phospholipids by phospholipase D.

When the major polar lipid of purple membrane, a dialkyl analogue of phosphatidyl glycerophosphate, is treated with phospholipase D under the usual assay conditions for this enzyme, the reaction yields dialkylglycerol and glycerol bisphosphate, i.e. the kind of products that would be expected from a phospholipase C reaction. The effect is seen both in native purple membranes and with the pure phospholipid in the form of liposomes. The specific activity and kinetic parameters Km and Vmax of phospholipase D for the purple membrane phospholipid are similar to those for egg phosphatidylcholine. The presence of phospholipase C impurities in the phospholipase D preparations has been ruled out as an explanation for the above observations. A hypothesis is suggested, taking into account the peculiar headgroup structure of the bacterial lipid, to explain the seemingly anomalous enzyme behavior.

Bacillus cereus↗
PubMed1990

The interaction of phospholipase A2 with phospholipid analogues and inhibitors.

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗
PubMed1990

Effects of phospholipase A2 on numbers of histamine H1 receptors in guinea pig lungs.

Effects of phospholipase A2 on numbers of histamine H1 receptors and muscarinic acetylcholine receptors were estimated in guinea pig lungs. Histamine H1 and muscarinic acetylcholine receptors in lung membranes were studied by the direct binding technique using 3H-pyrilamine and 3H-quinuclidinyl benzilate, respectively. The control group had two orders (high-affinity and low-affinity) of binding sites of histamine H1 receptors. Pretreatment of lung membranes with phospholipase A2 destroyed high-affinity binding sites, and these sites could not be detected after treatment. Low-affinity sites were not affected by the treatment. In contrast, the numbers of muscarinic acetylcholine receptors did not change significantly in spite of the addition of phospholipase A2. These results indicate that resistance of receptors against phospholipase A2 treatment varies among receptors and even between the binding sites in the same receptor. These variations might modulate pathological conditions associated with inflammation in which phospholipase is activated. Easy establishment of histamine tachyphylaxis might be explained by this mechanism.

Animals↗
PubMedDate not supplied

[Immobilization of phospholipase A2 from Central Asian cobra venom on polyamide sorbents].

The effect of the immobilization technique and the ligand nature on catalytic properties of phospholipase A2 from the cobra venom was studied. Preparations of phospholipase A2 adsorbed on and covalently bound to polyamide sorbents were obtained. The enzyme was coupled to polyamide beads modified with glutaraldehyde. In this case only 9% of the enzyme activity was retained. The enzyme adsorbed on polyamide modified with phosphatidylethanolamine retained up to 20% of the initial activity. The binding selectivity of phospholipase A2 was maximum in case of the sorbent with a binary ligand, e. g. phosphatidylethanolamine+cytotoxin, the sorbent capacity for the bound enzyme increased 2-3 times (460-600 units/g sorbent. The specific activity of the adsorbed phospholipase A2 was 17-40 units/g sorbent in contrast to 8.6 units/g sorbent for the covalently bound enzyme. Immobilization of the enzyme on polyamide sorbents resulted in changes of the pH-optimum, sensitivity to Ca2+ ions and the character of the enzyme-substrate interactions. Heart stability of the adsorbed phospholipase A2 was lower than that of the covalently bound enzyme. However, the adsorbed enzyme can be used, for example, in affinity chromatography due to its higher specific activity, selectivity and reversibility of the sorption.

Adsorption↗
PubMed1986

[Mechanism of stabilization of synaptosomes with alpha-tocopherol during exposure to phospholipase A2].

Changes in potential-dependent fluorescence were studied, using fluorescent probe di-S-C3-(5), in synaptosome suspensions exposed to phospholipase A2, alpha-tocopherol and its derivatives. Phospholipase A2 increased potential-dependent fluorescence, i.e. depolarization of synaptosome membranes. The damaging phospholipase A2 effect was prevented and/or abolished by alpha-tocopherol added to synaptosome suspensions before and after phospholipase A2. Alpha-tocopherol derivatives (2,2,5,7,8-pentamethyl-6-hydroxychromane and alpha-tocopheryl-acetate as well as 4-methyl-2,6-di-tert-butylphenol) failed to exert a protective effect on synaptosome membranes modified by phospholipase A2. It is suggested that alpha-tocopherol effect is determined by its interaction with fatty acids, with 6-hydroxy groups of chromanol nucleus and phytol chain being essential for the complex formation.

Animals↗
PubMed1986

Identification and purification of sheep platelet phospholipase A2 isoforms. Activation by physiologic concentrations of calcium ion.

Two families of platelet phospholipase A2 activity, were chromatographically resolved by anion exchange chromatography and were functionally distinguishable by their differential phospholipid subclass substrate specificity and calcium ion requirements. The major phospholipase A2 activity was present in the cytosolic compartment, eluted from DEAE-cellulose at 230 mM NaCl (hereafter referred to as phospholipase A2(beta)), and demonstrated a 100-fold selectivity in catalyzing the hydrolysis of 1-(O)-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmenylcholine) in comparisons with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (phosphatidylcholine). Phospholipase A2(beta) was purified to homogeneity by sequential gel filtration and Mono Q column chromatographies. Phospholipase A2(beta) eluted with an apparent molecular mass of 58 kDa during gel filtration chromatography and migrated as a single band with an apparent molecular mass of 30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that its native quaternary structure is dimeric. Fast protein liquid chromatography demonstrated that the polypeptides catalyzing this activity were comprised of multiple isoforms which possessed different specific activities. Each isoform required Ca2+ ion for activity and was completely activated over the range through which Ca2+ ion concentration is augmented in stimulated platelets (i.e. 300-800 nM).

Animals↗
PubMed1986

The phospholipase A1 of Trypanosoma brucei does not release myristate from the variant surface glycoprotein.

[3H]Myristoyl-labeled variant surface glycoprotein (VSG) has been isolated from Trypanosoma brucei by reverse phase high performance liquid chromatography and used as substrate for the conversion by trypanosomal enzymes of membrane-form VSG to soluble VSG. Conversion is detected by the release of myristoyl-containing lipids. The major lipolytic enzyme of T. brucei, phospholipase A1, is effective for the hydrolysis of myristoyl esters of p-nitrophenol, in a colorimetric assay. However, the phospholipase is unable to cleave the myristoyl ester linkage of VSG. The phospholipase can be separated from the myristoyl-releasing activity of trypanosome homogenate by centrifugation, affinity chromatography, and anion-exchange chromatography. Elution profiles on anion-exchange high performance liquid chromatography also indicate that the phospholipase is inactive against VSG. A small amount of myristoyl-releasing activity associated with the purified phospholipase is probably due to contamination with a phosphodiesterase which releases myristoyl-containing diglyceride from VSG.

Animals↗
PubMedDate not supplied

[Determination of phospholipase A2 and lysophospholipase A1 in a mixture].

The phospholipase A2 and lysophospholipase A1 activities were determined in various mixtures. When estimating the phospholipase A2 activity in venoms of snakes and insects the known methods based on measuring a change in the optical density of the egg yolk suspension are not acceptible if in investigated object besides phospholipases lysophospholipases are present (venoms of viper and giant hornet). In this case anomalous curves of changes in the system optical density in time are obtained. At the same time the analysis of such curves revealed that the method can be rather sensitive for determining lysophospholipases in venoms, as well as in phospholipase preparations of different purification, including electrophoretically homogeneous preparations. The results obtained enabled us to estimate the content of phospholipase A2 and lysophospholipase A1 in the venom of giant hornet and in its fractions, obtained during isolation and purification of the concerned enzymes.

Animals↗
PubMed1983

Release of phospholipase A2 activity from rabbit peritoneal neutrophils by f-Met-Leu-Phe.

Rabbit peritoneal neutrophils secrete phospholipase A2 activity in response to f-Met-Leu-Phe (FMLP). The secretion of phospholipase A2, like that of the known granule enzymes, requires cytochalasin B and is enhanced by extracellular Ca2+, and the time course of the release is rapid, being completed in less than a minute. The concentration dependence of the secretion of the phospholipase is the same as that of the known granule enzymes up to 10(-9) M FMLP. At this concentration, the release of the known granule enzymes reaches a maximum, but that of phospholipase A2 does not until 10(-8) M FMLP. The amount of enzyme activity released plus the amount of enzyme activity remaining in the pellet is distinctly greater than the total enzyme activity extractable from the cell before release. The phospholipase secreted is active in the neutral to alkaline range, requires Ca2+, is inhibited by low levels of detergent, and is of A2 specificity.

Animals↗
PubMed1983

[Simplified microdetermination of cerebral phospholipase A1, A2 and lysophopholipase].

The purpose of our study was to examine the ischemia induced enzymatic changes of decaylation-reacylation cycle of membrane phospholipids in dog brain. In this study, we developed new modified method for assay of phospholipase A1, A2 and lysophospholipase which is simpler and needs only a smaller amount of materials. For the first report, we introduced this new method and demonstrated some properties of phospholipase A1, A2 and lysophospholipase in dog brain. Crude enzyme solution for assays of phospholipase A1, A2 and lysophospholipase was gained from extraction of frozen brain with aceton, butanol and saline. The level of phosphorus in the enzyme extract was determined and only those extracts which had a level of phosphorus within a certain range were used. The substrates for assays were L-alpha-[beta-palmitoyl-1-14C] phosphatidylcholine, dipalmitoyl for phospholipase A1 and A2 and L-lysophosphatidylcholine-1-[1-14C] palmitoyl for lysophospolipase respectively. Each radioactive substrates was diluted with cold carrier lipid to give the proper specific activity. Reaction system including substrate, buffer [pH 7.0] and enzyme extract was incubated for 10 hours at 38 degrees C. But for the assay of phospholipase A1 and A2, enzyme solution was pre-incubated at 70 degrees C for 5 minutes. In our new method, reaction mixture was directly separated by TLC without extracting lipids. Enzyme activities were calculated from radio thin-layer chromatograms. Furthermore, we made a comparison between our method and the former one. The value of each enzyme activity was slightly higher in our method than in the former one. However, it was revealed that the results were reproducible in both methods.

Animals↗
PubMed1982

The role of phospholipase A2 lysines in phospholipolysis of Escherichia coli killed by a membrane-active neutrophil protein.

Purified rabbit bactericidal/permeability-increasing protein at bactericidal concentrations is a membrane-perturbing agent that triggers hydrolysis of envelope phospholipids of a phospholipase A-less Escherichia coli (S17) mutant by a highly basic (pI greater than 10) phospholipase A2, purified from Agkistrodon halys blomhoffii snake venom. Most other purified phospholipases A2 do not degrade the phospholipids of E. coli killed by the bactericidal protein. To study the role of enzyme charge in bactericidal protein-dependent phospholipid hydrolysis, lysines of the Agkistrodon phospholipase A2 were modified, either by carbamylation (decreases net charge), or by reductive methylation (no delta charge). Incorporation of [14C]cyanate or [14C]formaldehyde and amino acid analysis served to monitor modification. Modification appears to be limited to epsilon-NH2 groups. Incorporation of up to 5 mol of cyanate or formaldehyde/mol of enzyme did not affect catalytic activity. In contrast, incorporation of, on average, 1 mol of either reagent/mol of protein reduced by 80% the activity of the enzyme toward E. coli S17 killed by the bactericidal protein. Since this loss is similar with carbamylation and reductive methylation, the role of the epsilon-NH2 group in the bactericidal protein-dependent hydrolysis seems independent of charge. Thus, the lysines in this phospholipase A2 are not essential for catalysis and substrate binding, but are essential for the action of this enzyme on E. coli killed by the bactericidal protein.

Animals↗
PubMed1981

Premature labor. II. Bacterial sources of phospholipase.

Human term labor is thought to be initiated by amniotic and chorionic phospholipase A2, an enzyme that liberates arachidonic acid esters from the phospholipids of these membranes, leading to the synthesis of prostaglandins by the placental membranes. The striking association of premature labor with intrauterine infection or contamination, urinary tract infection, and early neonatal sepsis led us to study the microorganisms present in these infections for phospholipase A2 activity. Activity was found in Bacteroides fragilis, Peptostreptococcus, Fusobacterium necrophorum, Streptococcus viridans, Streptococcus fecalis, Streptococcus A and B, Escherichia coli, Klebsiella, Staphylococcus epidermidis, Pneumococcus, Lactobacillus, and Mycoplasma hominis. Bacteroides fragilis, Peptostreptococcus, Fusobacterium, and S viridans had the highest activities. The specific activities of phospholipase A2 from these organisms were several times higher than that of the membrane phospholipase A2 of the amnion and chorion. We postulate that premature labor may be initiated by microorganisms with phospholipase A2 activity from endocervical and/or intrauterine contamination or infection, producing deacylation of arachidonic acid from amniotic phospholipids with increased concentrations of free arachidonic acid and increased prostaglandin synthesis, which triggers labor.

Animals↗
PubMed1982

Identification of domains of phosphatidylcholine in human erythrocyte plasma membranes. Differential action of acidic and basic phospholipases A2 from Agkistrodon halys blomhoffii.

Highly purified acidic (pI 4.9) and basic (pI 8.7) phospholipases A2 from snake (Agkistrodon halys blomhoffii) venom hydrolyzed approximately 20% and 60%, respectively, of the phosphatidylcholine (PC) of intact human erythrocytes prior to hemolysis. Sequential use of the acidic enzyme followed by the basic phospholipase A2 or vice versa manifested a characteristic PC hydrolysis pattern. For example, when acidic enzyme had hydrolyzed nearly 20% of this substrate, a subsequent treatment with the basic enzyme hydrolyzed only an additional 40% of the PC before hemolysis. On the other hand, in experiments where hydrolysis of about 20% of PC of erythrocytes was achieved by a short term incubation with the basic enzyme, then a further treatment of the same cells with the acidic enzyme caused only 10% additional PC hydrolysis before hemolysis. This demonstrated that the acidic enzyme hydrolyzed one domain of PC in the intact erythrocytes, whereas the basic enzyme hydrolyzed not only the same but also another domain of PC in membranes. Analysis of fatty acids released by the action of these two phospholipases A2 on erythrocytes indicated further characteristic differences. In particular, the ratio of released saturated to unsaturated fatty acids was significantly higher with the acidic enzyme as compared with the basic phospholipase A2. These results provide firm support to the conclusion that there are different domains of PC in human erythrocyte membranes and that the acidic and basic phospholipase A2 of A. halys blomhoffii can be used to identify them.

Erythrocyte Membrane↗