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Journal of the American Chemical Society Mar 2018We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A specificity toward membrane phospholipid substrates. We discovered...
We demonstrate that lipidomics coupled with molecular dynamics reveal unique phospholipase A specificity toward membrane phospholipid substrates. We discovered unexpected headgroup and acyl-chain specificity for three major human phospholipases A. The differences between each enzyme's specificity, coupled with molecular dynamics-based structural and binding studies, revealed unique binding sites and interfacial surface binding moieties for each enzyme that explain the observed specificity at a hitherto inaccessible structural level. Surprisingly, we discovered that a unique hydrophobic binding site for the cleaved fatty acid dominates each enzyme's specificity rather than its catalytic residues and polar headgroup binding site. Molecular dynamics simulations revealed the optimal phospholipid binding mode leading to a detailed understanding of the preference of cytosolic phospholipase A for cleavage of proinflammatory arachidonic acid, calcium-independent phospholipase A, which is involved in membrane remodeling for cleavage of linoleic acid and for antibacterial secreted phospholipase A favoring linoleic acid, saturated fatty acids, and phosphatidylglycerol.
Topics: Binding Sites; Catalytic Domain; Humans; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Molecular Dynamics Simulation; Phospholipases A2, Calcium-Independent; Phospholipases A2, Cytosolic; Phospholipases A2, Secretory; Phospholipids; Substrate Specificity
PubMed: 29342349
DOI: 10.1021/jacs.7b12045 -
The Journal of Biological Chemistry Jul 1994Phospholipase A1 (PLA1) was isolated from a soluble fraction of bovine brain. The purification included sequential DEAE-Sephacel, phenyl-Sepharose FF, and...
Phospholipase A1 (PLA1) was isolated from a soluble fraction of bovine brain. The purification included sequential DEAE-Sephacel, phenyl-Sepharose FF, and heparin-Sepharose CL-6B column chromatography. Mono Q, Sephacryl S-300, and Mono S high resolution column chromatography in the presence of the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (10 mM) and glycerol (10%, v/v) was required to further separate the enzyme from contaminating material. The purified PLA1 eluted from the Sephacryl S-300HR column in a volume corresponding to a molecular mass of 365 kDa and migrated as two bands (M(r) = 112,000 and 95,000) when separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Chromatofocusing, hydroxylapatite, and lectin affinity column chromatography and nondenaturing polyacrylamide gel electrophoresis were unsuccessful in separating the two electrophoretic bands, implying a close association or similarity. The purified enzyme was stable in solutions containing detergent and glycerol and was insensitive to metal chelators, dithiothreitol, phenylmethylsulfonyl fluoride, and diisopropyl fluorophosphate, but was inactivated by heat (60 degrees C) and ZnCl2. At pH 7.5, the purified enzyme showed highest specific activity, 23.8 mumol/min-mg, when 1-palmitoyl-2-[1-14C]arachidonoyl-phosphatidylethanolamine was the substrate. The rate of catalysis was optimal at a pH of 9.0 and could be enhanced 2-fold by Ca2+, Mg2+, and Sr2+, but not Mn2+. The enzyme catalyzed the specific hydrolysis of acyl groups from the sn-1 position of a broad range of phospholipid substrates, including lysophospholipids, and accounts for most of the soluble phospholipase A1 activity of bovine brain.
Topics: Animals; Brain; Cattle; Cholic Acids; Chromatography; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Hydrogen-Ion Concentration; Phospholipases A; Phospholipases A1; Phospholipids; Salts; Substrate Specificity
PubMed: 8034719
DOI: No ID Found -
FASEB Journal : Official Publication of... Dec 2011Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs,...
Spinal cord injury (SCI) results in permanent loss of motor functions. A significant aspect of the tissue damage and functional loss may be preventable as it occurs, secondary to the trauma. We show that the phospholipase A(2) (PLA(2)) superfamily plays important roles in SCI. PLA(2) enzymes hydrolyze membrane glycerophospholipids to yield a free fatty acid and lysophospholipid. Some free fatty acids (arachidonic acid) give rise to eicosanoids that promote inflammation, while some lysophospholipids (lysophosphatidylcholine) cause demyelination. We show in a mouse model of SCI that two cytosolic forms [calcium-dependent PLA(2) group IVA (cPLA(2) GIVA) and calcium-independent PLA(2) group VIA (iPLA(2) GVIA)], and a secreted form [secreted PLA(2) group IIA (sPLA(2) GIIA)] are up-regulated. Using selective inhibitors and null mice, we show that these PLA(2)s play differing roles. cPLA(2) GIVA mediates protection, whereas sPLA(2) GIIA and, to a lesser extent, iPLA(2) GVIA are detrimental. Furthermore, completely blocking all three PLA(2)s worsens outcome, while the most beneficial effects are seen by partial inhibition of all three. The partial inhibitor enhances expression of cPLA(2) and mediates its beneficial effects via the prostaglandin EP1 receptor. These findings indicate that drugs that inhibit detrimental forms of PLA(2) (sPLA(2) and iPLA2) and up-regulate the protective form (cPLA2) may be useful for the treatment of SCI.
Topics: Animals; Enzyme Inhibitors; Female; Group II Phospholipases A2; Group IV Phospholipases A2; Group VI Phospholipases A2; Locomotion; Mice; Mice, Inbred BALB C; Mice, Knockout; Phospholipase A2 Inhibitors; Phospholipases A2; Receptor Cross-Talk; Receptors, Prostaglandin E, EP1 Subtype; Spinal Cord Injuries
PubMed: 21868473
DOI: 10.1096/fj.11-183186 -
Molecular Plant Pathology Sep 2023Phospholipase A (PLA ) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA (VlsPLA )...
Phospholipase A (PLA ) is a lipolytic enzyme that hydrolyses phospholipids in the cell membrane. In the present study, we investigated the role of secreted PLA (VlsPLA ) in Verticillium longisporum, a fungal phytopathogen that mostly infects plants belonging to the Brassicaceae family, causing severe annual yield loss worldwide. Expression of the VlsPLA gene, which encodes active PLA , is highly induced during the interaction of the fungus with the host plant Brassica napus. Heterologous expression of VlsPLA in Nicotiana benthamiana resulted in increased synthesis of certain phospholipids compared to plants in which enzymatically inactive PLA was expressed (VlsPLA ). Moreover, VlsPLA suppresses the hypersensitive response triggered by the Cf4/Avr4 complex, thereby suppressing the chitin-induced reactive oxygen species burst. VlsPLA -overexpressing V. longisporum strains showed increased virulence in Arabidopsis plants, and transcriptomic analysis of this fungal strain revealed that the induction of the gene contributed to increased virulence. VlsPLA was initially localized to the host nucleus and then translocated to the chloroplasts at later time points. In addition, VlsPLA bound to the vesicle-associated membrane protein A (VAMPA) and was transported to the nuclear membrane. In the nucleus, VlsPLA caused major alterations in the expression levels of genes encoding transcription factors and subtilisin-like proteases, which play a role in plant immunity. In conclusion, our study showed that VlsPLA acts as a virulence factor, possibly by hydrolysing host nuclear envelope phospholipids, which, through a signal transduction cascade, may suppress basal plant immune responses.
Topics: Virulence Factors; Phospholipases; Verticillium; Ascomycota; Arabidopsis; Plant Immunity; Plant Diseases
PubMed: 37171182
DOI: 10.1111/mpp.13352 -
The Journal of Biological Chemistry Sep 1985The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus... (Comparative Study)
Comparative Study
The kinetics of phospholipid hydrolysis by cobra venom phospholipase A2 were examined and compared to those of phospholipase A2 from porcine pancreas, Crotalus adamanteus (rattlesnake) venom, and bee venom. Only the enzyme from Naja naja naja (cobra) venom was found to be activated significantly by phosphorylcholine-containing compounds when hydrolyzing phosphatidylethanolamine. The cobra venom enzyme was also the only one in which these activators induced protein aggregation. The parallel specificity for activators and aggregators suggests that these two phenomena are linked. Product effects were also shown to vary between these four phospholipases. These effects manifest themselves in nonlinear time courses, in changes in steady state velocity, and in the differential effects of serum albumin on reaction rates. Different effects were even seen for the same enzyme when acting on different substrates. A model is presented to account for these observations; its main features are enzyme activation by an activator molecule, whose specificity depends on the enzyme, and an activator-induced aggregation of the enzyme.
Topics: Animals; Bee Venoms; Crotalid Venoms; Elapid Venoms; Enzyme Activation; Kinetics; Macromolecular Substances; Pancreas; Phospholipases; Phospholipases A; Phospholipases A2; Species Specificity; Swine
PubMed: 4030786
DOI: No ID Found -
Journal of Bacteriology Apr 1985The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions...
The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M). Phosphoglycerides labeled with [14C]palmitic acid and [3H]serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography. The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane. The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes. This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites. The highest phospholipase C activity was detected in the inner membrane and Int.M fractions. The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains.
Topics: Cell Membrane; Escherichia coli; Glycerophosphates; Lysophospholipase; Palmitic Acid; Palmitic Acids; Phosphatidylcholines; Phospholipases; Serine; Type C Phospholipases
PubMed: 3920208
DOI: 10.1128/jb.162.1.50-54.1985 -
The Journal of Biological Chemistry Jun 1997
Review
Topics: Animals; Calcium; Humans; Membrane Lipids; Phospholipases A; Phospholipases A2; Phospholipids
PubMed: 9195897
DOI: 10.1074/jbc.272.26.16069 -
Biochimica Et Biophysica Acta.... Oct 2021Inoculation of selected microbial species into the soils is one of the most effective means of bioremediation of soils polluted by persistent organic pollutants as well...
Interactions of fungal phospholipase Lecitase ultra with phospholipid Langmuir monolayers - Search for substrate specificity and structural factors affecting the activity of the enzyme.
Inoculation of selected microbial species into the soils is one of the most effective means of bioremediation of soils polluted by persistent organic pollutants as well as of biocontrol of plant pests. However, this procedure turns out frequently to be ineffective due to the membrane-destructive enzymes secreted to the soil by the autochthonous microorganisms. Especial role play here phospholipases and among them phospholipase A1 (PLA1), Therefore, to explain the interactions of microbial membranes and PLA1 at molecular level and to find the correlation between the composition of the membrane and its resistance to PLA1 action we applied phospholipid Langmuir monolayers as model microbial membranes. As a representative soil extracellular PLA1 we applied Lecitase ultra which is a commercially available hybrid enzyme of PLA1 activity. With the application of specific sn1-ether-sn2-ester phospholipids we proved that Lecitase ultra has solely PLA1 activity; thus, can be applied as an effective model of soil PLA1s. Our studies proved that this enzyme has vast substrate specificity and can hydrolyze structural phospholipids regardless the structure of their polar headgroup. It turned out that the hydrolysis rate was controlled by the condensation of the model membranes. These built of the phospholipids with long saturated fatty acid chains were especially resistant to the action of this enzyme, whereas these formed by the 1-saturated-2-unsaturated-sn-glycero-3-phospholipids were readily degraded. Regarding the polar headgroup we proposed the following row of substrate preference of Lecitase ultra: phosphatidylglycerols > phosphatidylcholines > phosphatidylethanolamines > cardiolipins.
Topics: Aspergillus oryzae; Hydrolysis; Phospholipases; Phosphorylation; Protein Conformation; Substrate Specificity
PubMed: 34175298
DOI: 10.1016/j.bbamem.2021.183687 -
Infection and Immunity Sep 1984The interaction of large numbers of viable Rickettsia prowazekii cells with L-cells results in the expression of a phospholipase A activity with the concomitant release...
The interaction of large numbers of viable Rickettsia prowazekii cells with L-cells results in the expression of a phospholipase A activity with the concomitant release of free fatty acids and lysophosphatides from the phospholipids of the L-cell. About 50% of rickettsiae present in the suspension that was centrifuged onto an L-cell monolayer at 0 degree C to effect this interaction formed a tight L-cell-rickettsiae association from which the rickettsiae could not be removed by simple washing. Both the L-cell-rickettsiae association and the rickettsiae before association with L-cells interact with N-ethylmaleimide, so that the subsequent expression of the phospholipase A activity was inhibited (treatment of the L-cells with N-ethylmaleimide before centrifugation does not inhibit phospholipase activity). However, treatment of this association with 2,4-dinitrophenol and KCN caused much less inhibition of this phospholipase A activity than did treatment of the rickettsiae with these agents before centrifugation onto the L-cells. Incubation of the L-cell-rickettsiae association for a short time at 35 degrees C resulted in a very low level of free fatty acid formation and changed this association to an activated complex in which the phospholipase A activity was no longer sensitive to the inhibitory effects of N-ethylmaleimide. The characteristics of the association and activated complex were stable: after a 2-h incubation at 0 degrees C, the association and the activated complex retained both their basal phospholipase A activities and their characteristic responses to N-ethylmaleimide treatment. In scanning electron micrographs of the activated complexes, the rickettsiae that were initially attached were no longer visible after 45 min at 35 degrees C, and the surface of the L-cell appeared to have been etched away. These activated complexes provide a system in which modulators of the phospholipase A can be investigated without the confusion caused by the first-step receptor interaction between rickettsiae and their host cells.
Topics: Animals; Ethylmaleimide; Mice; Microscopy, Electron, Scanning; Models, Biological; Phagocytosis; Phospholipases; Phospholipases A; Rickettsia prowazekii; Time Factors; Typhus, Epidemic Louse-Borne
PubMed: 6432695
DOI: 10.1128/iai.45.3.577-581.1984 -
The Journal of Antibiotics Jul 1979The effects of some antibiotics on activities of phospholipase A2, B and C were investigated in vitro. Tetracyclines, macrolides, chloramphenicol and carbenicillin...
The effects of some antibiotics on activities of phospholipase A2, B and C were investigated in vitro. Tetracyclines, macrolides, chloramphenicol and carbenicillin inhibited the activity of Crotalus adamanteus phospholipase A2 towards phospholipids of egg-yolk emulsions. When the ability to inhibit the activity of Penicillium notatum phospholipase B towards mixed micelles of phosphatidylcholine and Triton X-100 was investigated, polymyxin B was found to be inhibitory while chloramphenicol and carbenicillin were found to stimulate the activity of the phospholipase. The activity of Bacillus cereus phospholipase C towards the mixed micelles was inhibited by bleomycin, oleandomycin and chloramphenicol.
Topics: Carbenicillin; Chloramphenicol; Depression, Chemical; Lysophospholipase; Phospholipases; Phospholipases A; Phospholipases A2; Tetracyclines; Type C Phospholipases
PubMed: 541266
DOI: 10.7164/antibiotics.32.734