scholarly journals Phospholipase A2 from plasma of patients with septic shock is associated with high-density lipoproteins and C3 anaphylatoxin: some implications for its functional role

1995 ◽  
Vol 306 (1) ◽  
pp. 167-175 ◽  
Author(s):  
M A Gijón ◽  
C Pérez ◽  
E Méndez ◽  
M Sánchez Crespo
Keyword(s):  
Septic Shock ◽  
Enzyme Activity ◽  
Phospholipase A2 ◽  
Molecular Mass ◽  
Gel Filtration ◽  
High Density ◽  
Complement C3 ◽  
High Density Lipoproteins ◽  
Group Ii ◽  
Anaphylatoxin C3a

Phospholipase A2 (PLA2) activity was purified 12,544-fold with a 13% yield from the plasma of patients diagnosed of septic shock by the sequential use of heparin-agarose affinity chromatography, gel filtration, and reverse-phase f.p.l.c. Gel-filtration chromatography of plasma omitting high-ionic-strength buffer revealed a molecular mass different from that of purified PLA2 and co-elution with apolipoprotein A-I peaks, which suggests its association with high-density lipoproteins (HDL). N-terminal analysis of the enzyme activity protein band, electroblotted from a SDS-acrylamide gel and with an assessed molecular mass of 19 kDa, showed an identical sequence to that of alpha-chain of human C3 complement component, suggesting the presence in this band of a complex formed by a complement C3-derived anaphylatoxin (C3a)-related fragment and the PLA2 linked side-by-side. Because the preparation of plasma enzyme showed lower activity than the enzyme obtained from fibroblasts transfected with the coding sequence of human group-II PLA2, and because the addition of C3-derived anaphylatoxins from human serum inhibited the activity of this recombinant PLA2, it was considered that C3a-related peptides behave as inhibitors of group-II PLA2. The enzyme showed optimal activity on [14C]oleate-labelled autoclaved E. coli, on synthetic phosphatidylethanolamine, and on [3H]arachidonate-labelled membranes of the monoblast cell line U937, but it did not show any activity on the release of [3H]arachidonate from pre-labelled human polymorphonuclear leukocytes (PMNs). In short, PLA2 from plasma of sepsis patients shows unique associations with other plasma proteins which may influence its functional properties. The association with C3-related peptides shows an inhibitory effect on the enzyme activity, whereas the association with HDL might influence its environment and/or its interaction with cells. The study of the catalytic properties shows a prominent effect on bacterial phospholipids, synthetic phosphatidylethanolamine, and membranes from U937 monoblasts, but not on synthetic phosphatidylcholine or on PMNs, even when these cells were maintained in culture to allow spontaneous apoptosis and became a good substrate for pancreatic type PLA2.

Abstract 260: Knockout of Apolipoprotein A-II Has Dramatic Effects on High-Density Lipoprotein Subspecies Distribution

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Scott M Gordon ◽  
Catherine A Reardon ◽  
Godfrey S Getz ◽  
W S Davidson
Keyword(s):  
Gel Filtration ◽  
Density Lipoprotein ◽  
High Density ◽  
Paraoxonase 1 ◽  
High Density Lipoproteins ◽  
Ionization Mass ◽  
Associated Proteins ◽  
Compositional Diversity ◽  
The Impact ◽  
Modest Effect

High density lipoproteins (HDL) are a highly heterogeneous population of particles composed of various lipids and proteins. They have been demonstrated to possess a diverse variety of functional properties which are thought to contribute to protection against cardiovascular disease (CVD). Proteomics studies have identified up to 75 different proteins which can associate with HDL. The basis for the compositional diversity of HDL is not known but a better understanding will yield important information about its broad functional diversity. To investigate the impact of common HDL apolipoproteins on the distribution of other apolipoproteins, we have begun to systematically fractionate plasma from various HDL apolipoprotein KO mice. Plasma from apoA-I, apoA-IV and apoA-II global KO mice was applied to gel filtration chromatography to distinguish HDL size populations. HDL particles sequestered by a phospholipid binding resin were proteomically analyzed by electrospray ionization mass spectrometry. By comparing elution volume shifts (i.e. particle size variations) for each HDL protein between WT controls and the KO models, we assessed the impact of the deleted protein on HDL size distributions. Ablation of apoA-I, while decreasing total HDL phospholipid by 70%, had a surprisingly small impact on the distribution of the majority of other HDL associated proteins - affecting only 9 of them. Genetic apoA-IV ablation had a similar modest effect shifting a distinct subset of 9 proteins. However, loss of apoA-II, in addition to causing a similar 70% reduction in overall HDL phospholipids, affected the size distribution of some 45 HDL proteins (including several complement proteins and paraoxonase-1). These data suggest that apoA-I, while associated with the majority of HDL phospholipid, may actually interact with relatively few of the lower abundance proteins known to be associated with HDL. ApoA-II on the other hand, may interact with many of these, perhaps acting as a docking site or adaptor molecule.

scholarly journals Loss of sphingosine 1-phosphate (S1P) in septic shock is predominantly caused by decreased levels of high-density lipoproteins (HDL)

2019 ◽  
Vol 7 (1) ◽  
Author(s):  
Martin Sebastian Winkler ◽  
Konstantin B. Märtz ◽  
Axel Nierhaus ◽  
Günter Daum ◽  
Edzard Schwedhelm ◽  
...  
Keyword(s):  
Septic Shock ◽  
High Density ◽  
High Density Lipoproteins ◽  
Sphingosine 1 Phosphate

Changes in epitope exposition of apolipoprotein A-I on the surface of high density lipoproteins after phospholipase A2 treatment

1995 ◽  
Vol 117 (2) ◽  
pp. 159-167 ◽  
Author(s):  
Mario Menschikowski ◽  
Ute Hempel ◽  
Gerd Dinnebier ◽  
Peter Lattke ◽  
Klaus-Wolfgang Wenzel ◽  
...  
Keyword(s):  
Phospholipase A2 ◽  
High Density ◽  
High Density Lipoproteins ◽  
Apolipoprotein A

Hypoxia induces changes in phospholipase A2 in rat proximal tubules: evidence for multiple forms

1995 ◽  
Vol 269 (6) ◽  
pp. F846-F853 ◽  
Author(s):  
K. H. Choi ◽  
C. L. Edelstein ◽  
P. Gengaro ◽  
R. W. Schrier ◽  
R. A. Nemenoff
Keyword(s):  
Phospholipase A2 ◽  
Molecular Mass ◽  
Cell Injury ◽  
Gel Filtration ◽  
Proximal Tubules ◽  
Pla2 Activity ◽  
Extracellular Medium ◽  
Fatty Acid Release ◽  
Acid Release ◽  
Mass Form

Increased free fatty release during hypoxia is believed to contribute to cell injury. This phenomenon is likely to be mediated through activation of specific isoforms of phospholipase A2 (PLA2). In this study, PLA2 enzymatic activity was measured in cell-free extracts prepared from rat renal proximal tubules. Both soluble and membrane-associated PLA2 activity were detected. All PLA2 activity detected during normoxia was Ca2+ dependent. Fractionation of cytosolic extracts by gel filtration revealed three peaks of PLA2 activity. Exposure of tubules to hypoxia resulted in stable activation of soluble PLA2 activity, which correlated with disappearance of the highest molecular mass form (> 100 kDa) and appearance of a low-molecular-mass form (approximately 15 kDa) of PLA2. Hypoxia also resulted in release of a low-molecular-mass form of PLA2 into the extracellular medium. Pretreatment of tubules with glycine before hypoxia blocked this release of PLA2 but not activation of soluble PLA2 activity. These studies provide direct evidence for PLA2 activation during hypoxia and suggest that multiple mechanisms regulate free fatty acid release associated with hypoxia injury.

scholarly journals Affinity chromatography of human plasma low- and high-density lipoproteins. Elution by selective cleavage of a bond in the spacer

1979 ◽  
Vol 181 (3) ◽  
pp. 691-698 ◽  
Author(s):  
A Wichman
Keyword(s):  
Human Plasma ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Capacity ◽  
High Density ◽  
High Density Lipoproteins ◽  
Protein Solutions ◽  
Ester Bond ◽  
Triton X

Human plasma low- and high-density lipoproteins were found to bind to Sepharose gels containing coupled cholesterol or cholic acid. The lipoproteins were bound very strongly, and it was not possible to elute them under non-denaturing conditions. The detergents Triton X-100 and sodium dodecyl sulphate eluted the lipoproteins in partly denatured form. Adsorbents were used where the steroid was coupled through a spacer containing a thiol ester bond. It was thus possible to elute bound lipoproteins by selective cleavage of the bond with hydroxylamine. A small proportion of albumin was the only contaminant detected, the amounts depending on which ligand was used. Low- and high-density lipoproteins were separated by gel filtration. They behaved as did the native molecules when analysed by gel filtration, immunodiffusion, immunoelectrophoresis and electrophoresis in polyacrylamide gradient gels. The high capacity and the selectivity of the adsorbents make them suitable for the removal of lipoproteins from protein solutions.

Bronchoalveolar lavage fluid phospholipase A2 activities are increased in human adult respiratory distress syndrome

1995 ◽  
Vol 269 (1) ◽  
pp. L109-L118 ◽  
Author(s):  
D. K. Kim ◽  
T. Fukuda ◽  
B. T. Thompson ◽  
B. Cockrill ◽  
C. Hales ◽  
...  
Keyword(s):  
Lung Injury ◽  
Respiratory Distress Syndrome ◽  
Respiratory Distress ◽  
Bronchoalveolar Lavage ◽  
Phospholipase A2 ◽  
Molecular Mass ◽  
Adult Respiratory Distress Syndrome ◽  
Distress Syndrome ◽  
Pla2 Activity ◽  
Group Ii

The role of phospholipase A2 (PLA2) in lung injury in humans is unclear. Previous studies have failed to identify an increase in PLA2 activity in bronchoalveolar lavage fluids (BALF) of patients with the adult respiratory distress syndrome (ARDS). In this study, increased phospholipase A2 (PLA2) activity was detected in BALF from patients with ARDS. PLA2 levels in BALF correlated positively with lung injury score in patients with lung disease. BALF PLA2 activity in patients with ARDS was resolved into heparin binding and nonbinding activities. Both PLA2 activities were increased in BALF of ARDS patients. The PLA2 activity that bound to heparin was identified as a group II PLA2 by its chromatographic characteristics, its inhibition by dithiothreitol, its substrate specificity, and its approximate molecular mass of 14 kDa. The second PLA2 activity was further purified and found to require Ca2+ at a concentration > 2 x 10(-4) M for activity. This form of PLA2 exhibited a neutral and broad pH optimum (pH 6.0-8.0) and hydrolyzed both phosphatidylethanolamine and phosphatidylcholine effectively. Its apparent molecular mass was estimated to be 80–90 kDa. Neither anti-pancreatic PLA2 antiserum nor anti-pig spleen cytosolic 100-kDa PLA2 antiserum immunoprecipitated the enzymatic activity. Thus at least two forms of PLA2 are increased in activity in BALF of patients with ARDS, a group II PLA2 and a biochemically and immunochemically form distinct from group I, group II, and cytosolic PLA2. Increased lung PLA2 activity may be important for the pathophysiology of ARDS.

scholarly journals Effects of dexamethasone and transforming growth factor-β2 on group II phospholipase A2 mRNA and activity levels in interleukin 1β- and forskolin-stimulated mesangial cells

1996 ◽  
Vol 315 (2) ◽  
pp. 435-441 ◽  
Author(s):  
Margriet J. B. M. VERVOORDELDONK ◽  
Casper G. SCHALKWIJK ◽  
Josef PFEILSCHIFTER ◽  
Henk van den BOSCH
Keyword(s):  
Enzyme Activity ◽  
Growth Factor ◽  
Phospholipase A2 ◽  
Inflammatory Cytokines ◽  
Transforming Growth Factor ◽  
Mesangial Cells ◽  
Interleukin 1Β ◽  
Mrna Levels ◽  
Transforming Growth Factor Β2 ◽  
Group Ii

The expression of 14 kDa group II phospholipase A2 [also referred to as secretory PLA2 (sPLA2)] is induced in rat glomerular mesangial cells by exposure to inflammatory cytokines and forskolin, a cAMP elevating agent. Previously we have shown that dexamethasone and transforming growth factor-β2 (TGF-β2) suppress sPLA2 protein synthesis and enzyme activity induced by cytokines and forskolin. The regulation of sPLA2 by pro-inflammatory cytokines suggests that the enzyme may play a role in glomerular inflammatory reactions. In order to understand the regulation of sPLA2 in more detail, we investigated whether dexamethasone and TGF-β2 also suppress sPLA2 mRNA after its induction by either interleukin-1β (IL-1β) or forskolin. We found that IL-1β-induced sPLA2 mRNA in rat mesangial cells is not down-regulated by pretreatment of the cells with dexamethasone, even at a concentration of 10 μM, which dramatically decreases sPLA2 protein levels and activity. Metabolic labelling experiments indicated that the decreased sPLA2 levels under these conditions can be explained by inhibition of the rate of sPLA2 synthesis from the elevated mRNA levels. In contrast, the forskolin-induced elevation of sPLA2 mRNA is inhibited by dexamethasone in a concentration-dependent manner. Likewise, TGF-β2 inhibits the elevation of sPLA2 mRNAs induced by either IL-1β or forskolin. The decrease in sPLA2 mRNA caused by TGF-β2 corresponds with the decrease in sPLA2 enzyme levels and activity. These data suggest that cytokine- and forskolin-induced sPLA2 expression is tightly controlled via both transcriptional and post-transcriptional mechanisms. Furthermore, we show that pretreatment of mesangial cells with epidermal growth factor prior to stimulation with IL-1β or forskolin had no suppressing effect on sPLA2 levels or enzyme activity, as has been reported previously for osteoblasts.

scholarly journals Protein-Defined Subspecies of HDLs (High-Density Lipoproteins) and Differential Risk of Coronary Heart Disease in 4 Prospective Studies

2020 ◽  
Vol 40 (11) ◽  
pp. 2714-2727
Author(s):  
Frank M. Sacks ◽  
Liang Liang ◽  
Jeremy D. Furtado ◽  
Tianxi Cai ◽  
W. Sean Davidson ◽  
...  
Keyword(s):  
Coronary Heart Disease ◽  
Heart Disease ◽  
Relative Risk ◽  
Prospective Studies ◽  
Conditional Logistic Regression ◽  
Hazard Ratio ◽  
High Density ◽  
Apolipoprotein A1 ◽  
Complement C3 ◽  
High Density Lipoproteins

Objective: HDL (high-density lipoprotein) contains functional proteins that define single subspecies, each comprising 1% to 12% of the total HDL. We studied the differential association with coronary heart disease (CHD) of 15 such subspecies. Approach and Results: We measured plasma apoA1 (apolipoprotein A1) concentrations of 15 protein-defined HDL subspecies in 4 US-based prospective studies. Among participants without CVD at baseline, 932 developed CHD during 10 to 25 years. They were matched 1:1 to controls who did not experience CHD. In each cohort, hazard ratios for each subspecies were computed by conditional logistic regression and combined by meta-analysis. Higher levels of HDL subspecies containing alpha-2 macroglobulin, CoC3 (complement C3), HP (haptoglobin), or PLMG (plasminogen) were associated with higher relative risk compared with the HDL counterpart lacking the defining protein (hazard ratio range, 0.96–1.11 per 1 SD increase versus 0.73–0.81, respectively; P for heterogeneity <0.05). In contrast, HDL containing apoC1 or apoE were associated with lower relative risk compared with the counterpart (hazard ratio, 0.74; P =0.002 and 0.77, P =0.001, respectively). Conclusions: Several subspecies of HDL defined by single proteins that are involved in thrombosis, inflammation, immunity, and lipid metabolism are found in small fractions of total HDL and are associated with higher relative risk of CHD compared with HDL that lacks the defining protein. In contrast, HDL containing apoC1 or apoE are robustly associated with lower risk. The balance between beneficial and harmful subspecies in a person’s HDL sample may determine the risk of CHD pertaining to HDL and paths to treatment.

scholarly journals Rat liver mitochondrial phospholipase A2 is an endotoxin-stimulated membrane-associated enzyme of Kupffer cells which is released during liver perfusion

1993 ◽  
Vol 293 (1) ◽  
pp. 143-150 ◽  
Author(s):  
G M Hatch ◽  
D E Vance ◽  
D C Wilton
Keyword(s):  
Enzyme Activity ◽  
Phospholipase A2 ◽  
Rat Liver ◽  
Kupffer Cells ◽  
Hepatic Lipase ◽  
Liver Perfusion ◽  
Elevated Serum ◽  
Physiological Saline ◽  
Group Ii ◽  
Phospholipase A2 Activity

A novel fluorescence assay for phospholipase A2 [Wilton (1990) Biochem. J. 266, 435-439] has been used to study the Group-II rat liver mitochondrial enzyme, and a number of novel properties of this enzyme were identified. (1) The enzyme activity was located in the liver macrophages (Kupffer cells) while negligible activity was associated with hepatocytes. (2) Although subcellular fractionation of whole liver confirmed the predominantly mitochondrial location of this enzyme activity, the analysis of the hepatocyte-free Kupffer-cell-enriched fraction revealed a different enzyme distribution, with the majority of activity being associated with the microsomal membrane fraction. (3) Bacterial endotoxin has been previously shown to be scavenged by Kupffer cells in rats. Treatment of rats with bacterial lipopolysaccharide (endotoxin) resulted in a dramatic time- and dose-dependent increase in liver phospholipase A2 activity. (4) It is known that injection of endotoxin into rodents results in elevated serum phospholipase A2 activity, while a similar phenomenon is seen in the condition of septic shock in man. The source of this serum enzyme was unknown. In this study perfusion of livers from rats pretreated with lipopolysaccharide with physiological saline demonstrated a 6-fold increase in phospholipase A2 activity in the perfusate compared with sham-treated controls, with only minor release of hepatic lipase. (5) Western-blot analysis confirmed an increased release of this Group-II phospholipase A2 into the perfusate of lipopolysaccharide-treated rats compared with sham-treated controls. These results suggest that liver Kupffer cells are a major source of the endotoxin-induced serum Group-II phospholipase A2 activity associated with bacterial infection and trauma.

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