P3-340: PLA2 enzyme: New target for memantine action

2008 ◽  
Vol 4 ◽  
pp. T621-T621
Author(s):  
Fábio B. Mury ◽  
Nádia R. Barbosa ◽  
Patrícia P. Defillipo ◽  
Camila T. Mendes ◽  
Wagner F. Gattaz ◽  
...  
Keyword(s):  
Pla2 Enzyme

Highly Active Modified Variants of Recombinant Phospholipase А2 from Streptomyces violaceoruber for Effective Biosynthesis in Yeasts

2019 ◽  
pp. 30-41 ◽  
Author(s):  
E.P. Sannikova ◽  
A.V. Malysheva ◽  
F.A. Klebanov ◽  
D.G. Kozlov
Keyword(s):  
New Technologies ◽  
Specific Activity ◽  
Point Mutations ◽  
Egg Yolk ◽  
Enzyme Preparations ◽  
Highly Active ◽  
High Calcium ◽  
Pla2 Enzyme ◽  
Streptomyces Violaceoruber ◽  
The Cost

The capacity of yeast to produce the highly active variants of PLA2 has been confirmed. The high-active variants were based on the original enzyme from the strain А-2688 of Streptomyces violaceoruber. To reduce the enzyme toxicity and to increase its expression, various approaches were tested including point mutations, construction of artificial N- and/or C-end pro-regions, hybridization with other proteins and engineering or inactivation of glycosylation sites. As a main result, the modified PLA2 enzymes were obtained which have the same secretion level as their low-active predecessors, but specific activity of which was at least tenfold higher. As the main feature, the selected mutants were characterized by a lower affinity for Ca2+ that probably accounts for their low toxicity (and high expression capacity) at the stage of biosynthesis and their ability to activate under special conditions, e.g. during the egg yolk fermentation. The data obtained can provide a basis for the cost reduction of highly active PLA2 enzyme preparations in industries where the application of high calcium concentrations is allowed. recombinant phospholipase А2, Streptomyces violaceoruber, yeasts, secretion, producer strain The work was initiated by the Innovation Center Biriuch - New Technologies, Ltd., and was supported within the framework of the State Assignment no. 595-00004-18 PR.

scholarly journals Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D

2004 ◽  
Vol 380 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Yong-Xin SUN ◽  
Kazuhito TSUBOI ◽  
Yasuo OKAMOTO ◽  
Takeharu TONAI ◽  
Makoto MURAKAMI ◽  
...  
Keyword(s):  
Biosynthetic Pathway ◽  
Brain Homogenate ◽  
Wide Distribution ◽  
Rat Tissues ◽  
Lysophospholipase D ◽  
Pla2 Enzyme ◽  
First Time ◽  
Hydrolysis Of ◽  
The Brain

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2α were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

Elevated circulating levels of lipoprotein-associated phospholipase A2 in obese children

2015 ◽  
Vol 53 (7) ◽  
Author(s):  
Sophia Sakka ◽  
Tania Siahanidou ◽  
Chronis Voyatzis ◽  
Panagiota Pervanidou ◽  
Christina Kaminioti ◽  
...  
Keyword(s):  
Plasma Concentrations ◽  
Growth Curves ◽  
Normal Weight ◽  
Enzyme Linked Immunosorbent Assay ◽  
Z Score ◽  
Obese Children ◽  
Characteristic Analysis ◽  
Cvd Risk ◽  
Thromboembolic Risk ◽  
Pla2 Enzyme

AbstractObesity and cardiovascular disease (CVD) often co-exist, but the pathophysiologic mechanisms that link the two are not fully understood. Lipoprotein-associated phospholipase ASixty-seven lean [39 boys and 28 girls, mean body mass index (BMI) z-score –0.2±0.8] and 66 obese (32 boys and 34 girls, mean BMI z-score 4.4±1.2) age-matched (p=0.251) children, aged 6–12 years, were studied. BMI z-score was calculated based on the Greek BMI growth curves, and children were categorized as obese according to the Cole criteria. All children underwent physical examination and a fasting morning blood sample was obtained for glucose, insulin, lipid profile, and Lp-PLA2 assessment. Plasma concentrations of Lp-PLA2 were determined by a commercially available Lp-PLA2 enzyme-linked immunosorbent assay kit (PLAC Test), while other measurements were performed using standard methods.Plasma Lp-PLA2 levels were significantly higher in obese children (322.5±77.8 ng/mL) compared with normal-weight ones (278.0±64.4 ng/mL, p<0.001). Lp-PLA2 concentrations were significantly correlated with the BMI z-score (p=0.004). Receiver operating characteristic analysis on Lp-PLA2 values resulted in significant areas under the curve (AUC) for distinguishing between obese and normal-weight groups of children (AUC, 0.726; p<0.001).We found significantly higher Lp-PLA2 levels in obese children than lean controls. Interestingly, they all had levels >200 ng/mL, which are considered to correlate with atherosclerosis and a high thromboembolic risk in adults. The positive correlation of Lp-PLA2 with BMI suggests that Lp-PLA2 might be the link between obesity and increased cardiovascular risk, which can be elevated even at a very young age. Measurement of Lp-PLA2 in plasma could therefore represent a further biomarker for assessing increased CVD risk in obese children and adolescents.

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 Adeno-Associated Virus VP1u Exhibits Protease Activity

Viruses ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 399 ◽  
Author(s):  
Justin J. Kurian ◽  
Renuk Lakshmanan ◽  
William M. Chmely ◽  
Joshua A. Hull ◽  
Jennifer C. Yu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Protease Activity ◽  
Active Sites ◽  
Ethylenediaminetetraacetic Acid ◽  
Disordered Proteins ◽  
Group Iii ◽  
Monogenic Diseases ◽  
Pla2 Enzyme ◽  
Viral Protein 1

Adeno-associated viruses (AAVs) are being developed for gene delivery applications, with more than 100 ongoing clinical trials aimed at the treatment of monogenic diseases. In this study, the unique N-terminus of AAV capsid viral protein 1 (VP1u), containing a canonical group XIII PLA2 enzyme domain, was observed to also exhibit proteolytic activity. This protease activity can target casein and gelatin, two standard substrates used for testing protease function but does not self-cleave in the context of the capsid or target globular proteins, for example, bovine serum albumin (BSA). However, heated BSA is susceptible to VP1u-mediated cleavage, suggesting that disordered proteins are substrates for this protease function. The protease activity is partially inhibited by divalent cation chelators ethylenediaminetetraacetic acid (EDTA) and ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA), and human alpha-2-macroglobulin (A2M), a non-specific protease inhibitor. Interestingly, both the bovine pancreatic (group VIIA) and bee venom (group III) PLA2 enzymes also exhibit protease function against casein. This indicates that PLA2 groups, including VP1u, have a protease function. Amino acid substitution of the PLA2 catalytic motif (76HD/AN) in the AAV2 VP1u resulted in attenuation of protease activity, suggesting that the protease and PLA2 active sites are related. However, the amino acid substitution of histidine H38, which is not involved in PLA2 function, to alanine, also affects protease activity, suggesting that the active site/mechanism of the PLA2 and protease function are not identical.

scholarly journals Zinc and barium inhibit the phospholipase A2 from Naja naja atra by different mechanisms

1994 ◽  
Vol 301 (2) ◽  
pp. 503-508 ◽  
Author(s):  
M Mezna ◽  
T Ahmad ◽  
S Chettibi ◽  
D Drainas ◽  
A J Lawrence
Keyword(s):  
Phospholipase A2 ◽  
Binding Sites ◽  
Alternative Model ◽  
Competitive Inhibitor ◽  
Enzymic Activity ◽  
Crystallographic Analysis ◽  
Kinetic Characteristics ◽  
Naja Naja ◽  
Pla2 Enzyme ◽  
Naja Atra

The mode of inhibition of the phospholipase A2 (PLA2) enzyme from the Chinese cobra (Naja naja atra) by Zn2+ is qualitatively different from inhibition by Ba2+. Inhibition by Ba2+ shows the kinetic characteristics of a conventional competitive inhibitor acting to displace Ca2+ from a single essential site, but Zn2+ has the paradoxical property of being more inhibitory at high than at low Ca2+ concentration. Kinetic analysis of the Ca(2+)-dependence of enzymic activity shows a bimodal response, indicating the presence of two Ca(2+)-binding sites with affinities of 2.7 microM and 125 microM respectively, and we propose that these can be identified with the two Ca(2+)-binding sites revealed by crystallographic analysis [White, Scott, Otwinowski, Gleb and Sigler (1990) Science 250, 1560-1563]. The results are consistent with the model that the enzyme is activated by two Ca2+ ions, one that is essential and can be displaced by Ba2+, and one that modulates the activity by a further 5-10-fold and which can be displaced by Zn2+. An alternative model is also presented in which the modulating Zn(2+)-binding site is a phenomenon of the lipid/water interface.

scholarly journals Antisnake venom activity and isolation of quercetin from the leaf of Neocarya macrophylla (Sabine) Prance ex F. White (Malpighiales: Chrysobalanaceae)

2019 ◽  
Vol 6 (13) ◽  
pp. 381-389 ◽  
Author(s):  
A. J. Yusuf ◽  
M. I. Abdullahi ◽  
A. M. Musa ◽  
A. K. Haruna ◽  
V. Mzozoyana ◽  
...  
Keyword(s):  
Liquid Chromatography ◽  
Methanol Extract ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Scientific Basis ◽  
Traditional Treatment ◽  
Snake Envenomation ◽  
Nmr Data ◽  
Pla2 Enzyme ◽  
Vacuum Liquid Chromatography

Snake envenomation is a major cause of death and disability in many developing countries. Neocarya macrophylla (Sabine) Prance ex F. White (Malpighiales: Chrysobalanaceae) have been reportedly used in traditional medicine to treat snake envenomation. Bioassay-guided isolation of antivenom principles was carried out on the leaf of N. macrophylla against Naja nigricollis venom. The methanol extract of N. macrophylla leaf and its ethylacetate and n-butanol fraction significantly (P < 0.05) protected mice against venom-induced lethality with 100% survival rate and there was remarkable inhibition of the poisonous effects of PLA2 enzyme by the extracts and the fractions. Encouraged by this result, the ethylacetate soluble fraction was subjected to purification using vacuum liquid chromatography and gel filtration which led to the isolation of quercetin as the bioactive principle. The identity of the compound was determined on the basis of chemical tests, and by comparison of its 1H-NMR data with literature, this is the first report of isolation of this compound from the leaf of the plant. However, the results of the study suggests that the leaf of N. macrophylla possess significant antisnake venom activity which provide the scientific basis for its use in traditional treatment of snakebites.

scholarly journals PLASMEPSIN INHIBITORS; IS PLA2 ENZYME THE NATURAL INHIBITOR IN HUMANS AGAINST PLASMEPSINS PRODUCED BY MALARIAL PARASITE IN THEIR ERYTHROCYTIC CYCLE?

2019 ◽  
Vol 8 (5) ◽  
Author(s):  
Manoj G Tyagi
Keyword(s):  
Red Blood Cells ◽  
Aspartic Acid ◽  
Blood Cells ◽  
Host Cells ◽  
Malarial Parasite ◽  
Plasmodium Parasite ◽  
Malarial Infection ◽  
Erythrocytic Cycle ◽  
Pla2 Enzyme ◽  
Natural Inhibitor

Malaria is a mosquito-borne infectious disease that affects humans and other animals. Most deaths in malarial infection are caused by P. falciparum because P. vivax, P. ovale, and P. malariae generally cause a milder form of malaria. Within the red blood cells, the parasites multiply further, again asexually, periodically breaking out of their host cells to invade fresh red blood cells. Several such amplification cycles occur. Thus, classical descriptions of waves of fever arise from simultaneous waves of merozoites escaping and infecting red blood cells. Plasmepsin is a hemoglobin-degrading enzyme produced by the plasmodium parasite. It is an aspartic acid protease having 2 aspartic acid residues in the active site. On the other hand phospholipase A2 levels are increased in malarial infection and this may possibly provide protection against the effects of plasmepsin. This review examines the importance of this enzyme and interaction with plasmepsin.

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