Assembly of an intact Golgi complex requires phospholipase A2 (PLA2) activity, membrane tubules, and dynein-mediated microtubule transport

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.

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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

Biochemical Journal ◽

10.1042/bj2500343 ◽

1988 ◽

Vol 250 (2) ◽

pp. 343-348 ◽

Cited By ~ 17

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.

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Cerebrospinal Fluid Secretory Ca2+-Dependent Phospholipase A2 Activity Is Increased in Alzheimer Disease

Clinical Chemistry ◽

10.1373/clinchem.2009.130286 ◽

2009 ◽

Vol 55 (12) ◽

pp. 2171-2179 ◽

Cited By ~ 36

Author(s):

Sonia Chalbot ◽

Henrik Zetterberg ◽

Kaj Blennow ◽

Tormod Fladby ◽

Inge Grundke-Iqbal ◽

...

Keyword(s):

Cerebrospinal Fluid ◽

Alzheimer Disease ◽

Phospholipase A2 ◽

Microtiter Plate ◽

Pla2 Activity ◽

Activity Assay ◽

Proinflammatory Mediators ◽

Spla2 Activity ◽

Phospholipase A2 Activity ◽

Microtiter Plate Format

Abstract Background: The phospholipase A2 (PLA2) family comprises multiple isoenzymes that vary in their physicochemical properties, cellular localizations, calcium sensitivities, and substrate specificities. Despite these differences, PLA2s share the ability to catalyze the synthesis of the precursors of the proinflammatory mediators. To investigate the potential of PLA2 as a biomarker in screening neuroinflammatory disorders in both clinical and research settings, we developed a PLA2 assay and determined the predominant types of PLA2 activity in cerebrospinal fluid (CSF). Methods: We used liposomes composed of a fluorescent probe (bis-Bodipy® FL C11-PC [1,2-bis-(4,4- difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine]) and 1,2-dioleoyl-l-α-phosphatidylcholine as a substrate to measure CSF PLA2 activity in a 96-well microtiter plate format. We established the type of CSF PLA2 activity using type-specific inhibitors of PLA2. Results: Using 5 μL CSF per assay, our PLA2 activity assay was reproducible with CVs <15% in 2 CSF samples and for recombinant secretory Ca2+-dependent PLA2 (sPLA2) in concentrations ranging from 0.25 to 1 μmol/L. This PLA2 assay allowed identification of sPLA2 activity in lumbar CSF from healthy individuals 20–77 years old that did not depend on either sex or age. Additionally, CSF sPLA2 activity was found to be increased (P = 0.0008) in patients with Alzheimer disease. Conclusions: Adult human CSF has sPLA2 activity that can be measured reliably with the assay described. This enzyme activity in the CSF is independent of both sex and age and might serve as a valuable biomarker of neuroinflammation, as we demonstrated in Alzheimer disease.

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Comparison of Lipoprotein-Associated Phospholipase A2 and High Sensitive C-Reactive Protein as Determinants of Metabolic Syndrome in Subjects without Coronary Heart Disease: In Search of the Best Predictor

International Journal of Endocrinology ◽

10.1155/2015/934681 ◽

2015 ◽

Vol 2015 ◽

pp. 1-7 ◽

Cited By ~ 10

Author(s):

Mónica Acevedo ◽

Paola Varleta ◽

Verónica Kramer ◽

Giovanna Valentino ◽

Teresa Quiroga ◽

...

Keyword(s):

Metabolic Syndrome ◽

Phospholipase A2 ◽

High Sensitivity ◽

Roc Curves ◽

Activity Levels ◽

Pla2 Activity ◽

C Reactive Protein ◽

Cross Sectional ◽

Reactive Protein ◽

Cv Disease

High sensitivity C-reactive protein (hsCRP) is a marker of metabolic syndrome (MS) and cardiovascular (CV) disease. Lipoprotein-associated phospholipase A2 (Lp-PLA2) also predicts CV disease. There are no reports comparing these markers as predictors of MS.Methods. Cross-sectional study comparing Lp-PLA2 and hsCRP as predictors of MS in asymptomatic subjects was carried out; 152 subjects without known atherosclerosis participated. Data were collected on demographics, cardiovascular risk factors, anthropometric and biochemical measurements, and hsCRP and Lp-PLA2 activity levels. A logistic regression analysis was performed with each biomarker and receiver operating characteristic (ROC) curves were constructed for MS.Results. Mean age was 46 ± 11 years, and 38% of the subjects had MS. Mean Lp-PLA2 activity was 185 ± 48 nmol/mL/min, and mean hsCRP was 2.1 ± 2.2 mg/L. Subjects with MS had significantly higher levels of Lp-PLA2 (P=0.03) and hsCRP (P<0.0001) than those without MS. ROC curves showed that both markers predicted MS.Conclusion. Lp-PLA2 and hsCRP are elevated in subjects with MS. Both biomarkers were independent and significant predictors for MS, emphasizing the role of inflammation in MS. Further research is necessary to determine if inflammation predicts a higher risk for CV events in MS subjects.

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Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan: Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase

Biochemical Journal ◽

10.1042/bj3260867 ◽

1997 ◽

Vol 326 (3) ◽

pp. 867-876 ◽

Cited By ~ 60

Author(s):

Inbal HAZAN ◽

Raya DANA ◽

Yoseph GRANOT ◽

Rachel LEVY

Keyword(s):

Protein Kinase ◽

Nadph Oxidase ◽

Phospholipase A2 ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Cytosolic Phospholipase A2 ◽

Human Neutrophils ◽

Pla2 Activity ◽

Cytosolic Phospholipase ◽

Gf 109203X

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibitor genistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.

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

10.1055/s-0038-1644628 ◽

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.

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Performance Characteristics and Clinical Value of the Lipoprotein-Associated Phospholipase A2 by an Enzymatic Kinetic Method

Laboratory Medicine ◽

10.1093/labmed/lmy086 ◽

2019 ◽

Vol 50 (3) ◽

pp. 273-278

Author(s):

Mengli Lu ◽

Liying Lv

Keyword(s):

Risk Factors ◽

Phospholipase A2 ◽

Kinetic Method ◽

Reference Interval ◽

Reference Intervals ◽

Performance Characteristics ◽

Pla2 Activity ◽

Clinical Value ◽

Traditional Risk Factors ◽

Assay Precision

Abstract Objective To analyze the performance characteristics, stability, and clinical value of lipoprotein-associated phospholipase A2 (Lp-PLA2) using an enzymatic kinetic method. Methods The performance characteristics included reference intervals, precision, and accuracy. We assessed Lp-PLA2 stability by comparing Lp-PLA2 changes under different conditions. Lp-PLA2 was determined in the following groups: control individuals, patients with coronary heart disease (CHD), patients of different lipid subgroups within CHD, and patients with high total cholesterol (TC). Also, correlations between Lp-PLA2 and traditional cardiovascular risk factors were assessed. Results The mean (SD) reference interval of serum Lp-PLA2 activity was 451 (113) U per L with sex differences. Inter- and intra-assay precision revealed coefficients of variance (CVs) of 1.81% to 2.63% and 1.43% to 1.77%. The average bias was 0.33%. Lp-PLA2 activity was stable. In the CHD group, high-lipid subgroups, and high-TC group, Lp-PLA2 was elevated, and correlation was observed between Lp-PLA2 and traditional risk factors. Conclusion Lp-PLA2 activity has important clinical value in CHD.

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Characterization of phospholipase A2 (PLA2) activity in gerbil brain: enhanced activities of cytosolic, mitochondrial, and microsomal forms after ischemia and reperfusion

Journal of Neuroscience ◽

10.1523/jneurosci.11-06-01829.1991 ◽

1991 ◽

Vol 11 (6) ◽

pp. 1829-1836 ◽

Cited By ~ 111

Author(s):

G Rordorf ◽

Y Uemura ◽

JV Bonventre

Keyword(s):

Phospholipase A2 ◽

Ischemia And Reperfusion ◽

Pla2 Activity ◽

Gerbil Brain

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ARACHIDONATE METABOLISM IN RAT PLATELETS IN THE PRESENCE OF Ca2+-SUBSTITUTES (Sr2+, BA2+)

10.1055/s-0038-1643392 ◽

1987 ◽

Author(s):

D BLACHE ◽

M CIAVATTI

Keyword(s):

Phospholipase A2 ◽

Direct Evidence ◽

Optimum Concentration ◽

Activation Process ◽

Pla2 Activity ◽

Platelet Activity ◽

Subsequent Formation ◽

Lipoxygenase Products ◽

Subsequent Activation ◽

Rat Platelets

To further document the involvement of external Ca2+ in the platelet induced-activation process, we have studied the arachidonate (AA) metabolism of intact washed rat platelets in the presence of different concentrations of Ca2+, Sr2+ or Ba2+. The thrombin-induced mobilization of [3H]- or [14C]-AA preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products (CLP). Results indicate that upon thrombin (<0.1 U/ml) stimulation, AA release and CLP formation are reduced by 50 to 90% only by omission of Ca2+ as compared to 1 mM Ca2+ in the suspending medium. In higher Ca2+ concentrations (5 mM), the AA mobilization and CLP formation are inhibited and the data are close to those obtained in the absence of Ca2+. In the presence of Sr2+ or Ba2+, results indicate that these cations can substitute to Ca2+. Similarly to Ca2+, an optimum concentration is found in Sr2+ or Ba2+ (3-5 mM) and higher concentrations inhibit AA release and CLP formation.We have also studied the activity of a semi-purified preparation of phospholipase A2 (PLA2) from rat platelets. This activity was assayed (pH 9) using heat-denaturated (3H)-AA prelabeled phospholipids as substrate. The results show that this PLA2 activity was strongly Ca2+-dependent. We found that unlike Mg2+, Sr2+ and Ba2+ are able to greatly enhance this activity. Relative effiency (Vmax) are in the order Ca2+>Sr2+>Ba2+. Taken together, these findings as well as our previous results (Blache et al, BBA 1987, in press) suggest that external Ca2+ may play a major role in the regulation of rat platelet activity. Our interpretation is in line with the view that Sr2+ or Ba2+ could enter the platelet through a mechanism common to Ca2+ (a kind of Ca2+ channel). Although direct evidence await the results of further studies which are in progress, it can be considered that Sr2+ or Ba2+ might cause platelet induced-activation mimickigci a rise in the cytosolic Ca2+ and subsequent activation of Ca2+-dependent enzymes.

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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

Biochemical Journal ◽

10.1042/bj2940261 ◽

1993 ◽

Vol 294 (1) ◽

pp. 261-270 ◽

Cited By ~ 36

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.

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