ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE

1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith
Keyword(s):  
Phospholipase A2 ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Human Platelets ◽  
Phospholipid Bilayer ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Enzyme Substrate ◽  
Membrane Bound ◽  
Layer Chromatography

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.

scholarly journals Modulation of phospholipase A2 activity by the tumour promoters phorbol esters and teleocidin

1990 ◽  
Vol 268 (1) ◽  
pp. 169-173 ◽  
Author(s):  
K Y Nam ◽  
A Morino ◽  
S Kimura ◽  
H Fujiki ◽  
Y Imanishi
Keyword(s):  
Phospholipase A2 ◽  
Membrane Structure ◽  
Phorbol Esters ◽  
Hydrolytic Activity ◽  
Phospholipid Bilayer ◽  
Structural Defects ◽  
Moderate Degree ◽  
Pla2 Activity ◽  
Tumour Promoters ◽  
High Concentrations

The effects of tumour promoters, namely phorbol esters and teleocidin, on the activity of porcine pancreatic phospholipase A2 (PLA2) was investigated by using a system of small unilamellar vesicles composed of dipalmitoyl-phosphatidylcholine (DPPC). DPPC vesicles encapsulating Quin 2 (Quin 2/DPPC vesicles) were suspended in a medium containing Ca2+. The addition of PLA2 to Quin 2/DPPC vesicles increased the fluorescence intensity of Quin 2. This increase was due to chelation of Quin 2 with Ca2+, which resulted from an increase in the permeability of the phospholipid bilayer caused by the hydrolytic activity of PLA2. The tumour promoters phorbol 12-myristate 13-acetate (PMA) and teleocidin, at low concentrations, enhanced PLA2 activity at temperatures below the phase-transition temperature of the membrane, but, in contrast, high concentrations of the tumour promoters suppressed PLA2 activity. Phorbol 12-myristate (PM) also had a similar effect on PLA2 activity. PMA and PM disturbed the membrane structure markedly, which was indicated by the enhanced leakage of carboxyfluorescein (CF) from DPPC vesicles encapsulating CF. On the other hand, phorbol 12,13-didecanoate and 4 alpha-phorbol 12,13-didecanoate, which did not disturb the membrane structure to the same extent, had an insignificant effect on PLA2 activity. It is therefore concluded that PLA2 catalyses the hydrolysis of phospholipids in bilayer vesicles which contain a moderate degree of structural defects. However, the effects of tumour promoters on PLA2 activity was not related to their potencies as inflammatory and tumour-promoting agents.

scholarly journals Demonstration of calcium-dependent phospholipase A2 activity in membrane preparation of rabbit neutrophils. Absence of activation by fMet-Leu-Phe, phorbol 12-myristate 13-acetate and A-kinase

1988 ◽  
Vol 250 (2) ◽  
pp. 343-348 ◽  
Author(s):  
T Matsumoto ◽  
W Tao ◽  
R I Sha'afi
Keyword(s):  
Arachidonic Acid ◽  
Cyclic Amp ◽  
Phospholipase A2 ◽  
Kinase Inhibitor ◽  
Calcium Ionophore ◽  
Membrane Preparation ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Pla2 Activity ◽  
Intact Cells

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.

Changes In Arachidonate-Containing Complex Lipid During Platelet Stimulation

1981 ◽  
Author(s):  
S Rittenhouse-Simmons
Keyword(s):  
Human Platelets ◽  
Ionophore A23187 ◽  
Inhibitory Effects ◽  
Rapid Changes ◽  
Complex Lipid ◽  
Lipid Metabolites ◽  
Layer Chromatography ◽  
Sufficient Stimulus ◽  
Rate Of Response ◽  
Stimulation Of

We have examined changes in the 3H-arachidonic acid (3HC20:4) content of phospholipid following stimulation of human platelets by thrombin, ionophore A23187, or collagen. Resolution of lipids and lipid metabolites was achieved using HPLC and thin-layer chromatography after extraction of platelets by a modified Bligh and Dyer technique. The most rapid changes, leading to formation of TXB2, occur in response to thrombin and A23187. Both stimuli induce marked losses of 3HC20:4 from phosphatidylcholine (PC) and phosphatidylinositol (PI) within 30 sec. Collagen also induces losses in PI and PC and formation of diglyceride (DG), which become appreciable only after 1-3 min. Whereas thrombin and A23187 exhibit similarities in terms of the rate of response, these agents differ with regard to the phospholipases activated. Exposure of platelets to A23187 causes some activation of Ca+2-dependent PI-specific phospholipase C (PI-PLC); however, A23187 is an inefficient stimulus for this enzyme. Ionophore induces the formation of 1/4 to 1/6 as much PI- PLC-derived DG as does thrombin when comparable amounts of PI are hydrolyzed. A similar discrepancy is found with respect to generation of phosphatidic acid. Simultaneous addition of A23187 and thrombin does not impair the formation and metabolism of DG promoted by thrombin alone. Further, whereas indcmethacin (1-100 μg/ml) exerts no inhibitory effects on PLC or the formation of DG in stimulated platelets, indomethacin does inhibit 1) the loss of 3HC20:4 from PC in response to thrombin and 2) the loss of 3HC20:4 from PC and PI in response to A23187. Indomethacin has been reported to inhibit platelet phospholipase A2. It is clear from these studies that the activation of PI-PLC is not triggered merely by a general Ca+2 flux. In contrast, a shift in platelet Ca+2 stores appears to be a sufficient stimulus for phospholipase A.

scholarly journals Purification of a 100 kDa phospholipase A2 from spleen, lung and kidney: antiserum raised to pig spleen phospholipase A2 recognizes a similar form in bovine lung, kidney and platelets, and immunoprecipitates phospholipase A2 activity

1993 ◽  
Vol 294 (1) ◽  
pp. 261-270 ◽  
Author(s):  
D K Kim ◽  
J V Bonventre
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase A2 ◽  
Large Scale ◽  
Biochemical Characterization ◽  
Tissue Injury ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Pla2 Activity ◽  
Purification Scheme ◽  
Pla2 Enzyme

Phospholipase A2 (PLA2) plays a key role in the production of intracellular and extracellular chemical mediators such as arachidonic acid, eicosanoids and platelet-activating factor, which modulate membrane channel activity, signal transduction, are vasoactive and chemotactic, and are implicated in many pathophysiological mechanisms of inflammation and tissue injury. We previously identified, purified and characterized an arachidonic acid-selective cytosolic 100-110 kDa PLA2 from bovine platelets and rat kidney that is activated during cell stimulation. The purification schemes previously published resulted in low yields of enzyme, insufficient for extensive biochemical characterization. We report the purification of a large-molecular-mass (100 kDa) PLA2 from pig spleen, bovine kidney and bovine lung, using a novel large-scale purification scheme. The enzyme was purified to near homogeneity from an acidified extract obtained from 4.8 kg of pig spleen by sequential use of DEAE-cellulose anionic exchange, Butyl-Toyopearl hydrophobic chromatography and DEAE-5PW h.p.l.c., and further purified by non-denaturing PAGE. This purification scheme will permit the preparation of quantities of purified native enzyme sufficient to study its properties and regulation. To generate antiserum against the PLA2 enzyme, the 100 kDa protein was excised and electroeluted from SDS/PAGE gels of the active fractions after DEAE-5PW h.p.l.c., and this was used as antigen. This polyclonal antibody against pig spleen 100 kDa PLA2 protein reacted with 100 kDa bands in preparations partially purified from bovine platelets, kidney and lung as well as pig spleen, and immunoprecipitated PLA2 activity from these sources. The antibody also immunoprecipitated a 100 kDa protein from cytosolic fractions of cultured renal mesangial cells, human erythroleukaemia cells and human monocytic U937 cells. Considerable PLA2 activity was present in the immunoprecipitates. To our knowledge this antibody is unique in its ability to permit measurement of PLA2 activity in the immunoprecipitate itself, and will be a useful tool for the study of the regulation and the activation mechanisms of the native PLA2 enzyme.

scholarly journals Role of a membrane-associated serine esterase in the oxidant activation of phospholipase A2 by t-butyl hydroperoxide

1993 ◽  
Vol 292 (2) ◽  
pp. 585-589 ◽  
Author(s):  
S Chakraborti ◽  
J R Michael ◽  
G H Gurtner ◽  
S S Ghosh ◽  
G Dutta ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Membrane ◽  
Phospholipase A2 ◽  
Antioxidant Vitamin ◽  
Serine Esterase ◽  
Pla2 Activity ◽  
Butyl Hydroperoxide ◽  
Synthetic Substrate ◽  
Esterase Inhibitors ◽  
Membrane Bound

Exposure of bovine pulmonary-arterial endothelial cells to the oxidant lipid t-butyl hydroperoxide (t-Bu-OOH) increases cell-membrane-associated phospholipase A2 (PLA2) activity and stimulates arachidonic acid (AA) release. To test the hypothesis that a membrane-associated serine esterase plays an important role in activating PLA2, the present study was undertaken. In addition to increasing PLA2 activity and AA release, t-Bu-OOH also enhances the activity of a membrane-associated serine esterase that cleaves the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester (TAME). Changes in the activity of this membrane-bound serine esterase correlate directly with changes in the activity of PLA2. Serine esterase inhibitors such as phenylmethanesulphonyl fluoride, di-isopropyl fluorophosphate and alpha 1-proteinase inhibitor, and TAME, a synthetic substrate for serine esterase, prevent the increase in serine esterase activity, PLA2 activity and AA release caused by t-Bu-OOH. Pretreatment of the endothelial cells with the antioxidant vitamin E prevents t-Bu-OOH-induced stimulation of AA release and the cell-membrane-associated serine esterase and PLA2 activities. Adding t-Bu-OOH or the serine esterase trypsin to the endothelial-cell membrane fraction also significantly augments PLA2 activity, implying that these treatments activate latent PLA2. These results suggest that t-Bu-OOH stimulates a membrane-associated serine esterase that plays a crucial role in activating PLA2 and releasing AA.

Role of serine esterase in A23187-mediated activation of phospholipase A2 in pulmonary endothelium

1993 ◽  
Vol 264 (6) ◽  
pp. L538-L542 ◽  
Author(s):  
S. Chakraborti ◽  
J. R. Michael
Keyword(s):  
Endothelial Cell ◽  
Cell Membrane ◽  
Phospholipase A2 ◽  
Esterase Activity ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Serine Esterase ◽  
Pla2 Activity ◽  
Pulmonary Endothelium

To test the hypothesis that an endothelial cell membrane-associated serine esterase is involved in regulating phospholipase A2 (PLA2), we studied the effect of the calcium ionophore A23187 on intracellular PLA2 activity and arachidonic acid (AA) release in bovine pulmonary arterial endothelial cells. Exposure of these cells to A23187 causes a concentration-dependent increase in PLA2 activity and [14C]AA release. In addition to increasing PLA2 activity and AA release, A23187 enhances the activity of endothelial cell membrane-associated serine esterase that acts on the synthetic substrate N alpha-p-tosyl-L-arginine methyl ester. Serine esterase inhibitors, such as phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate, prevent the A23187-mediated increase in serine esterase activity, PLA2 activity, and AA release. Pretreatment of the cells with actinomycin D or cycloheximide does not prevent the A23187-mediated increase in AA release, serine esterase activity, or PLA2 activity. The membrane-associated serine esterase activity directly correlates with membrane PLA2 activity. These results suggest that a membrane-associated serine esterase plays a pivotal role in regulating PLA2 activity after exposure to A23187.

scholarly journals Phospholipase A2 enzyme from the venom of Egyptian honey bee Apis mellifera lamarckii with anti-platelet aggregation and anti-coagulation activities

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Doaa A. Darwish ◽  
Hassan M. M. Masoud ◽  
Mohamed M. Abdel-Monsef ◽  
Mohamed S. Helmy ◽  
Hind A. Zidan ◽  
...  
Keyword(s):  
Apis Mellifera ◽  
Platelet Aggregation ◽  
Phospholipase A2 ◽  
Honey Bee ◽  
Specific Activity ◽  
Deae Cellulose ◽  
High Specific Activity ◽  
Inhibitory Effect ◽  
Bee Venom ◽  
Very High

Abstract Background Honey bee venom contains various enzymes with wide medical and pharmaceutical applications. Results The phospholipase A2 (PLA2) has been apparently purified from the venom of Egyptian honey bee (Apis mellifera lamarckii) 8.9-fold to a very high specific activity of 6033 U/mg protein using DEAE–cellulose and Sephacryl S-300 columns. The purified bee venom PLA2 is monomeric 16 kDa protein and has isoelectric point (pI) of 5.9. The optimal activity of bee venom PLA2 was attained at pH 8 and 45 °C. Cu2+, Ni2+, Fe2+, Ca2+, and Co2+ exhibited a complete activating effect on it, while Zn2+, Mn2+, NaN3, PMSF, N-Methylmaleimide, and EDTA have inhibitory effect. Conclusions The purified bee venom PLA2 exhibited anti-platelet aggregation and anti-coagulation activities which makes it promising agent for developing novel anti-clot formation drugs in future.

RELEASE AND TRANSACYLATION OF ARACHIDONATE FROM A COMMON POOL OF 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE IN HUMAN PLATELETS

1987 ◽  
Author(s):  
A D Purdon ◽  
J B Smith
Keyword(s):  
Molecular Species ◽  
Specific Activity ◽  
Gel Filtration ◽  
Human Platelets ◽  
Time Interval ◽  
Resting Cells ◽  
Ether Phospholipid ◽  
Before And After ◽  
Layer Chromatography ◽  
Derivatives Of

We have previously shown that the main source of arachidonate in thrombin-stimulated human platelets is 1-acyl-2-arachidonoyl (AA) glycerophosphocholine (GPC) and release of 3H-AA from this phospholipid also was correlated with increased 3H-AA in ether phospholipid. This ATP independent transfer of 3H-AA from 1,2 diacyl GPC to ether phospholipid (transacylation) also occurs in resting cells. Human platelets in 1/10 volume of plasma (ACD anticoagulant, pH 6.5) were radiolabelled with 3H-AA for 60 min at 37°C and then exogenous 3H-AA was removed by gel filtration into Tyrode's buffer, pH 7.4, 0.2% albumin. These radiolabelled cells were incubated in the absence of exogenous 3H-AA for four hours followed by Bligh and Dyer extraction and thin layer chromatography purification of phospholipids. 3H-AA in 1,2 diacyl GPC was found to decrease by over 20% and increase substantially in 1-0-alkyl-2-acyl GPC and 1-0-alk-1'-enyl-2-acyl glycerophospho ethanolamine (GPE), In this same time interval the mass of AA released by thrombin (5 U/ml, 10 min, 37°C, no stirring)in the presence of BIT 775C and measured by GLC, stayed the same (30 nmoles/109 cells), however, the specific activity decreased. Using reverse phase HPLC to resolve diradylglycerobenzoate derivatives of phospholipids: acylation, deacylation, and transacylation were observed for individual AA-containing molecular species of phospholipid, including those with an unsaturated fatty acid at sn-1. In particular the radiolabellinq of the 1-unsaturate-2-arachidonoyl GPC correlated with the specific activity of the 3H-AA released by stimulation with thrombin. Furthermore, 1-arachidonoyl-2-3H-arachidonoyl GPC was completely deacylated while 50 % of its mass remained. This contrasted with 16:0, and 18:0-2-arachidonoyl GPC in which the specific activity remained the same before and after deacylation. We conclude that deacylation of AA-containing molecular species of 1,2 diacyl GPC in stimulated cells includes molecular species which are also a source of arachidonic acid for transacylation reactions.

scholarly journals Activation of human platelet phospholipase C by ionophore A23187 is totally dependent upon cyclo-oxygenase products and ADP

1984 ◽  
Vol 222 (1) ◽  
pp. 103-110 ◽  
Author(s):  
S E Rittenhouse
Keyword(s):  
Arachidonic Acid ◽  
Phosphatidic Acid ◽  
Phospholipase A2 ◽  
Phospholipase C ◽  
Human Platelet ◽  
Human Platelets ◽  
Dense Granule ◽  
Ionophore A23187 ◽  
Free Arachidonic Acid ◽  
Stimulation Of

Human platelets exposed to the Ca2+ ionophore A23187 form cyclo-oxygenase metabolites from liberated arachidonic acid and secrete dense granule substituents such as ADP. I have shown previously that A23187 causes activation of phospholipase A2 and some stimulation of phospholipase C. I now report that, in contrast to the case for thrombin, the activation of phospholipase C in response to ionophore is completely dependent upon the formation of cyclo-oxygenase products and the presence of ADP. The addition of A23187 to human platelets induces a transient drop in the amount of phosphatidylinositol 4,5-bisphosphate, a decrease in the amount of phosphatidylinositol, and the formation of diacylglycerol and phosphatidic acid. In addition, lysophosphatidylinositol and free arachidonic acid are produced. The presence of cyclo-oxygenase inhibitors or agents which remove ADP partially impairs these changes. When both types of inhibitor are present, the changes in phosphatidylinositol 4,5-bisphosphate and the formation of diacylglycerol and phosphatidic acid are blocked entirely, whereas formation of lysophosphatidylinositol and free arachidonic acid are relatively unaffected. The prostaglandin H2 analogue U46619 activates phospholipase C. This stimulation is inhibited partially by competitors for ADP. I conclude that phospholipase C is not activated by Ca2+ in the platelet, and suggest that stimulation is totally dependent upon a receptor coupled event.

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