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1.
Curr Drug Deliv ; 17(9): 806-814, 2020.
Article in English | MEDLINE | ID: mdl-32735519

ABSTRACT

AIM: This study aimed to investigate the existence of phospholipase-A (PLA) activity in Soluble L. major Antigens (SLA) because of no reports for it so far. Liposomes were used as sensors to evaluate PLA activity. OBJECTIVES: Liposomal SLA consisting of Egg Phosphatidylcholine (EPC) or Sphingomyelin (SM) were prepared by two different methods in different pH or temperatures and characterized by Dynamic Light Scattering (DLS) and Thin Layer Chromatography (TLC). METHODS: Lipid hydrolysis led to the disruption of EPC liposomal SLA in both methods but the Film Method (FM) produced more stable liposomes than the Detergent Removal Method (DRM). RESULT: The preparation of EPC liposomal SLA at pH 6 via FM protected liposomes from hydrolysis to some extent for a short time. EPC liposomes but not SM liposomes were disrupted in the presence of SLA. CONCLUSION: Therefore, a phospholipid without ester bond such as SM should be utilized in liposome formulations containing PLA as an encapsulating protein.


Subject(s)
Leishmania major/enzymology , Leishmaniasis Vaccines/chemistry , Leishmaniasis, Cutaneous/prevention & control , Phospholipases A/metabolism , Protozoan Proteins/chemistry , Adjuvants, Immunologic/administration & dosage , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/metabolism , Antigens, Protozoan/administration & dosage , Antigens, Protozoan/chemistry , Antigens, Protozoan/metabolism , Drug Compounding/methods , Drug Stability , Enzyme Assays , Humans , Hydrogen-Ion Concentration , Hydrolysis , Leishmania major/immunology , Leishmaniasis Vaccines/administration & dosage , Leishmaniasis Vaccines/metabolism , Leishmaniasis, Cutaneous/immunology , Leishmaniasis, Cutaneous/parasitology , Liposomes/chemistry , Liposomes/metabolism , Phosphatidylcholines/administration & dosage , Phosphatidylcholines/metabolism , Phospholipases A/isolation & purification , Protozoan Proteins/administration & dosage , Protozoan Proteins/metabolism , Sphingomyelins/administration & dosage , Sphingomyelins/metabolism
2.
Photochem Photobiol Sci ; 13(11): 1561-7, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25232894

ABSTRACT

The prominent local myotoxic effects induced by Bothrops snake venom are due, in part, to myotoxins. This effect is not neutralized by antivenom, which is the main therapy for victims of snakebite. Two basic myotoxins named MjTX-I and MjTX-II were isolated from Bothrops moojeni venom. Both myotoxins have a Lys-49 phospholipase A2 structure devoid of enzymatic activity, but are highly myonecrotic and edema-inducing. In this study, we analyzed the effect of a low-level laser (LLL) at 685 nm, an energy density of 2.2 J cm(-2), and the irradiation time of 15 s, and a light emitting diode (LED) at 635 or 945 nm at energy densities of 4 and 3.8 J cm(-2), and irradiation times of 41 and 38 s, respectively, applied 30 min and 3 h after edema formation in mice caused by MjTX-I or MjTX-II. MjTX-I or MjTX-II caused a significant edema formation in envenomed paws. LLL and LED irradiation significantly reduced the edema formation by both myotoxins from 1 up to 6 hours after the injection. Both LLL and LEDs were similar in reducing the edema formation induced by myotoxins. The combined photobiostimulation with antivenom had the same effect in reducing edema as treatment with the LLL or LEDs alone. In conclusion, the results of this study indicate that photobiostimulation could be used in association with antivenom therapy for treatment of local effects of Bothrops species venom.


Subject(s)
Bothrops/metabolism , Edema/chemically induced , Phospholipases A/toxicity , Venoms/metabolism , Animals , Edema/radiotherapy , Low-Level Light Therapy , Male , Mice , Phospholipases A/isolation & purification , Phospholipases A/metabolism
3.
Plant Physiol ; 162(1): 39-51, 2013 May.
Article in English | MEDLINE | ID: mdl-23542150

ABSTRACT

The release of fatty acids from membrane lipids has been implicated in various metabolic and physiological processes, but in many cases, the enzymes involved and their functions in plants remain unclear. Patatin-related phospholipase As (pPLAs) constitute a major family of acyl-hydrolyzing enzymes in plants. Here, we show that pPLAIIIδ promotes the production of triacylglycerols with 20- and 22-carbon fatty acids in Arabidopsis (Arabidopsis thaliana). Of the four pPLAIIIs (α, ß, γ, δ), only pPLAIIIδ gene knockout results in a decrease in seed oil content, and pPLAIIIδ is most highly expressed in developing embryos. The overexpression of pPLAIIIδ increases the content of triacylglycerol and 20- and 22-carbon fatty acids in seeds with a corresponding decrease in 18-carbon fatty acids. Several genes in the glycerolipid biosynthetic pathways are up-regulated in pPLAIIIδ-overexpressing siliques. pPLAIIIδ hydrolyzes phosphatidylcholine and also acyl-coenzyme A to release fatty acids. pPLAIIIδ-overexpressing plants have a lower level, whereas pPLAIIIδ knockout plants have a higher level, of acyl-coenzyme A than the wild type. Whereas seed yield decreases in transgenic plants that ubiquitously overexpress pPLAIIIδ, seed-specific overexpression of pPLAIIIδ increases seed oil content without any detrimental effect on overall seed yield. These results indicate that pPLAIIIδ-mediated phospholipid turnover plays a role in fatty acid remodeling and glycerolipid production.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Fatty Acids/metabolism , Phospholipases A/metabolism , Phospholipids/metabolism , Plant Oils/metabolism , Seeds/enzymology , Acyl Coenzyme A/analysis , Acyl Coenzyme A/metabolism , Arabidopsis/cytology , Arabidopsis/genetics , Arabidopsis Proteins/genetics , Arabidopsis Proteins/isolation & purification , Fatty Acids/analysis , Gene Expression , Gene Expression Regulation, Plant , Gene Knockout Techniques , Mutation , Organ Specificity , Phosphatidylcholines/metabolism , Phospholipases A/genetics , Phospholipases A/isolation & purification , Plant Oils/analysis , Plants, Genetically Modified , RNA, Messenger/genetics , RNA, Plant/genetics , Seeds/cytology , Seeds/genetics , Triglycerides/analysis , Triglycerides/metabolism , Up-Regulation
4.
Eukaryot Cell ; 10(6): 776-81, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21478434

ABSTRACT

Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels.


Subject(s)
Lipid Metabolism , Phospholipases A/metabolism , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Amino Acid Motifs/genetics , Amino Acid Sequence , Enzyme Assays , Esterases/metabolism , Gene Knockout Techniques , Membrane Proteins/metabolism , Molecular Sequence Data , Organelle Size/genetics , Organelles/metabolism , Organelles/ultrastructure , Phospholipases A/genetics , Phospholipases A/isolation & purification , Protein Transport/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Saccharomyces cerevisiae/ultrastructure , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/isolation & purification , Sequence Alignment
5.
Basic Clin Pharmacol Toxicol ; 104(4): 293-9, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19320636

ABSTRACT

We genetically modified Eclipta alba using Agrobacterium rhizogenes LBA 9402, with the aim of producing secondary metabolites with pharmacological properties against phospholipase A(2) and the myotoxic activities of snake venom. Extracts from in natura aerial parts and roots, both native and genetically modified (in vitro), were prepared and analysed by high-performance liquid chromatography. In natura materials showed the coumestan wedelolactone at higher concentration in the aerial parts, while demethylwedelolactone appeared at higher concentration in roots. Among the modified roots, clone 19 showed higher concentrations of these coumestans. Our results show that the in natura extracts of plants collected from Botucatu and Ribeirão Preto were efficient in inhibiting snake venom phospholipase A(2) activity. Regarding in vitro material, the best effect against Crotalus durissus terrificus venom was that of clone 19. Clone 19 and isolated coumestans (wedelolactone and demethylwedelolactone) inhibited the myotoxic activity induced by basic phospholipases A(2) isolated from the venoms of Crotalus durissus terrificus (CB) and Bothrops jararacussu (BthTX-I and II). The search for antivenom is justified by the need of finding active principles that are more efficient in neutralizing snake venoms and also as an attempt to complement serum therapy.


Subject(s)
Crotalid Venoms/antagonists & inhibitors , Eclipta/chemistry , Phospholipases A/antagonists & inhibitors , Plant Extracts/pharmacology , Animals , Bothrops , Brazil , Coumarins/isolation & purification , Coumarins/pharmacology , Crotalid Venoms/enzymology , Crotalus , Eclipta/genetics , Male , Mice , Phospholipases A/isolation & purification , Plant Components, Aerial , Plant Extracts/genetics , Plant Roots , Plants, Genetically Modified/chemistry , Rhizobium/genetics
6.
Toxicon ; 51(8): 1509-19, 2008 Jun 15.
Article in English | MEDLINE | ID: mdl-18501940

ABSTRACT

BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.


Subject(s)
Bothrops , Crotalid Venoms/chemistry , Neurotoxins/chemistry , Phospholipases A/chemistry , Amino Acid Sequence , Animals , Chemical Fractionation , Chickens/physiology , Chromatography, High Pressure Liquid , Crotalid Venoms/enzymology , Crotalid Venoms/pharmacology , Crotalus/metabolism , Crotoxin/isolation & purification , Crotoxin/pharmacology , Kinetics , Male , Mice , Molecular Sequence Data , Neuromuscular Blockade , Neurotoxins/isolation & purification , Neurotoxins/pharmacology , Phospholipases A/isolation & purification , Phospholipases A/pharmacology , Sequence Alignment , Sequence Analysis, Protein , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
7.
Artif Organs ; 31(5): 395-401, 2007 May.
Article in English | MEDLINE | ID: mdl-17470210

ABSTRACT

Continuous hemodiafiltration (CHDF) has been performed for the treatment of severe acute pancreatitis. Phospholipase A2 (PLA2) is one of the important mediators which exacerbate acute pancreatitis, but whether PLA2 can be removed by CHDF is unclear. In this study, the kinetics of group IB and group IIA PLA2 was examined at the first session of low-volume CHDF in eight patients with severe acute pancreatitis. CHDF was performed using polysulfone hemofilters (surface area: 0.7 m(2)) at a blood flow rate of 100 mL/min and a filtration and dialysate flow rate of 10 mL/min each. The plasma concentrations of group IB and IIA PLA2 before the start of CHDF were 47.4 +/- 52.0 microg/L and 352 +/- 390 microg/L, respectively, and did not change significantly. The clearances of group IB and IIA PLA2 achieved by the CHDF circuit 1 h after the start of CHDF were 20.7 +/- 11.6 mL/min and 16.7 +/- 4.4 mL/min, respectively, with both clearances decreasing significantly with time. The clearance of group IB PLA2 into the waste fluid tended to increase with time; however, the concentrations of group IIA PLA2 in the waste fluid were less than the measurable sensitivity. These results indicate that group IB PLA2 is adsorbed on the hemofilter membrane in preference to being removed into the waste fluid, while group IIA PLA2 is mainly removed by adsorption. However, low-volume CHDF is not effective at eliminating the group IB and IIA PLA2 plasma concentration.


Subject(s)
Hemodiafiltration/methods , Pancreatitis/therapy , Phospholipases A/blood , APACHE , Adsorption , Adult , Aged , Female , Group IB Phospholipases A2 , Group II Phospholipases A2 , Humans , Kinetics , Male , Middle Aged , Pancreatitis/blood , Phospholipases A/isolation & purification , Phospholipases A2 , Prospective Studies
8.
Curr Top Med Chem ; 7(8): 801-9, 2007.
Article in English | MEDLINE | ID: mdl-17456043

ABSTRACT

Ursolic acid (3beta-hydroxy-urs-12-en-28-oic acid) isolated from many medicinal plants has diverse pharmacologically important properties, including strong anti-inflammatory activity. However its interaction with pro-inflammatory PLA2 is not known. Ursolic acid inhibited secretory PLA2 (sPLA2) enzymes purified from Vipera russelli, Naja naja venom and human pleural fluid and synovial fluid. IC50 values determined for these enzymes ranged from 12 to 18 microM. Group II secretory PLA2 from both venoms & human inflammatory source were found to be sensitive to inhibition in comparison with group I cobra venom sPLA2. Variation in Ca2+ concentration from 2.5-15 mM did not alter the level of inhibition. Similarly sPLA2 inhibition by ursolic acid is independent of substrate concentration. Ursolic acid interacts with purified venom sPLA2 enzymes and enhances relative fluorescence intensity in a dose dependent manner. In the presence of ursolic acid apparent shift in the far UV-CD spectra of sPLA2 was observed, indicating a direct interaction with the enzyme and formation of enzyme-ursolic acid complex. This complex results in irreversible inhibition of sPLA2 as evident by dialysis study. Inhibition of sPLA2 induced mouse paw edema and indirect hemolytic activity confirmed its sPLA2 inhibitory activity in vivo and in situ respectively. These studies revealed that the strong anti-inflammatory activity of ursolic acid is by inhibiting sPLA2 enzymes.


Subject(s)
Enzyme Inhibitors/therapeutic use , Phospholipases A/antagonists & inhibitors , Triterpenes/pharmacology , Animals , Anti-Inflammatory Agents/pharmacology , Calcium/pharmacology , Edema/drug therapy , Group II Phospholipases A2 , Hemolysis/drug effects , Humans , Inhibitory Concentration 50 , Mice , Phospholipases A/isolation & purification , Phospholipases A2 , Pleural Cavity/enzymology , Snake Venoms , Spectrum Analysis , Synovial Fluid/enzymology , Triterpenes/therapeutic use , Ursolic Acid
9.
J Bacteriol ; 189(11): 4153-60, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17416658

ABSTRACT

We describe mycobacterial phospholipase A activity (MPLA) and, using reverse genetics, have associated this activity with putative mycobacterial cutinase. PLAs, which hydrolyze fatty acids on phospholipids, play a significant role in human inflammatory states and disease pathogenesis. In prokaryotes, the recognition of their role in virulence is more recent. Cutinases are serine esterases whose primary substrate is cutin, the waxy exterior layer of plants. Mycobacterium tuberculosis has maintained seven putative cutinases, though it should not encounter cutin; we demonstrate that known cutinases and MPLA cleave phospholipids in a PLA-type manner and also hydrolyze Tween. We analyzed cutinase motifs in mycobacteria and found the motif very prevalent. All mycobacteria tested had MPLA activity. These studies suggest an alternative use for putative cutinases by the M. tuberculosis group that is likely related to MPLA activity and lipid metabolism.


Subject(s)
Carboxylic Ester Hydrolases/metabolism , Mycobacterium/enzymology , Phospholipases A/metabolism , Amino Acid Sequence , Carboxylic Ester Hydrolases/genetics , Carboxylic Ester Hydrolases/isolation & purification , Chromatography, Thin Layer , Hydrolysis , Kinetics , Membrane Lipids/metabolism , Molecular Sequence Data , Mycobacterium/genetics , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Nitrobenzenes/metabolism , Phospholipases A/genetics , Phospholipases A/isolation & purification , Polysorbates/metabolism , Sequence Homology, Amino Acid
10.
Peptides ; 28(5): 969-73, 2007 May.
Article in English | MEDLINE | ID: mdl-17383773

ABSTRACT

Group IIA secretory phospholipases A(2) (sPLA(2)-II) is generally known to display potent gram-positive bactericidal activity, while group IA sPLA(2) (sPLA(2)-I) reportedly is not. In this work, a novel sPLA(2)-I named BFPA was identified from Bungarus fasciatus venom, and its antimicrobial activity was studied as well. The amino acid sequence of the venomous protein precursor was 145-amino acid in length, and contained a predicted 27-amino acid signal peptide and a 118-amino acid mature protein. Unlike the well-known sPLA(2)-Is, which have 14 half-cysteines forming 7 intramolecular disulfide bridges, BFPA possesses 15 half-cysteines. The additional cysteine might contribute to the formation of an intermolecular disulfide bridge of the homodimeric protein. In the biological activities assays, BFPA displayed the activities of anticoagulation and bactericidal against Escherichia coli and Staphylococcus aureus. This study is the first report about gram-positive bactericidal activity of sPLA(2)-I.


Subject(s)
Anti-Bacterial Agents/isolation & purification , Bungarus/metabolism , Elapid Venoms/enzymology , Phospholipases A/isolation & purification , Amino Acid Sequence , Animals , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Base Sequence , Blood Coagulation/drug effects , Chromatography, Gel , Chromatography, High Pressure Liquid , Dimerization , Dose-Response Relationship, Drug , Electrophoresis, Polyacrylamide Gel , Escherichia coli/drug effects , Group IV Phospholipases A2 , Molecular Sequence Data , Molecular Weight , Phospholipases A/chemistry , Phospholipases A/pharmacology , Sequence Alignment , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Staphylococcus aureus/drug effects
11.
Biotechnol Bioeng ; 98(1): 48-59, 2007 Sep 01.
Article in English | MEDLINE | ID: mdl-17318911

ABSTRACT

Phospholipases A(2) (PLA(2)) play an important role for the production of lysophospholipids. Presently they are mainly obtained from porcine or bovine pancreas but these mammalian sources are not accepted in several fields of application. To make accessible a non-mammalian PLA(2) to industrial application, synthetic genes encoding PLA(2) from honey bee (Apis mellifera) with modified N-termini were constructed and expressed in Escherichia coli. While expression of the gene with an N-terminal leader sequence to direct the protein into the periplasm failed, four variants with slightly modified N-termini (I1A-PLA(2), I1V-PLA(2), His(6)-tagged PLA(2) and PLA(2) still containing the start methionine) were successfully expressed. In all cases, the PLA(2) variants were produced as inclusion bodies. Their protein content amounted to 26-35% of total cell protein. The optimized renaturation procedure and subsequent purification by cation-exchange chromatography yielded pure active enzymes in yields of 4-11 mg L(-1). The recombinant PLA(2) variants showed activities, far-UV CD and fluorescence spectra similar to the glycosylated PLA(2) isolated from the venom glands of honey bee (bv-PLA(2)). The thermodynamic stabilities of the recombinant enzymes calculated from the transition curves of guanidine hydrochloride induced unfolding were also nearly identical to the stability of bv-PLA(2). For the variant I1A-PLA(2) high-cell density fermentation in 10 L-scale using mineral salt medium was shown to increase the volumetric enzyme yield considerably.


Subject(s)
Bees/enzymology , Bees/genetics , Escherichia coli/metabolism , Phospholipases A/biosynthesis , Phospholipases A/chemistry , Protein Engineering/methods , Animals , Enzyme Activation , Enzyme Stability , Escherichia coli/genetics , Industrial Microbiology/methods , Phospholipases A/genetics , Phospholipases A/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Substrate Specificity
12.
Biochim Biophys Acta ; 1770(4): 585-93, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17270350

ABSTRACT

BaTX PLA(2), a K49 phospholipase A(2) homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on mu-Bondapack C-18. A molecular mass of 13898.71 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that BaTX has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The complete amino acid sequence of BaTX PLA(2) contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence was SLFELGKMIL QETGKNPAKS YGAYYCYCGW GGQGQPKDAT DRCCYVHKCC YKKLTGCNPK KDRYSYSWKD KTIVCGENNS CLKELCECDK AVAICLRENL NTYNKKYRYY LKPLCKKADA C. In mice, BaTX induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. The LD(50) of BaTX was 7 mug/g body weight, by intravenous route. In vitro, the toxin caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparations. The blockage 50% was achieved at a concentration of 0.03 microM: 40+/-0.4 min and 0.07 microM: 35+/-0.3 min. Moreover, this protein induced a rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. Thus, the combined structural and functional information obtained identify BaTX as a new member of the K49 PLA(2) family, which presents the typical bioactivities described for such proteins.


Subject(s)
Bothrops/metabolism , Crotalid Venoms/enzymology , Phospholipases A/chemistry , Phospholipases A/toxicity , Amino Acid Sequence , Animals , Cell Line , Cell Survival/drug effects , Chickens , Chromatography, Gel , Chromatography, High Pressure Liquid , Dose-Response Relationship, Drug , Edema/chemically induced , In Vitro Techniques , Isoenzymes/chemistry , Lethal Dose 50 , Lysine , Mice , Molecular Sequence Data , Molecular Weight , Muscle, Skeletal/drug effects , Muscle, Skeletal/pathology , Myoblasts, Skeletal/drug effects , Necrosis , Neuromuscular Junction/drug effects , Phospholipases A/isolation & purification , Phospholipases A2 , Protein Conformation , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Time Factors
13.
Protein J ; 26(1): 39-49, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17203396

ABSTRACT

Cdr-12 and Cdr-13 isoforms of PLA2, a D49 protein, were purified from Crotalus durissus ruruima venom after one chromatographic step, reverse phase HPLC on micro-Bondapack C-18. The molecular mass by SDS-PAGE of Cdr-12 and Cdr-13 isoforms of PLA2 was 14333.49 Da and 14296.42 Da, respectively and confirmed by MALDI-TOF mass spectrometry. The amino acid composition showed that both isoforms Cdr-12 and Cdr-13 have a high content of Lys, Tyr, Gly, Arg, and 14 half-Cys residues, typical of a basic PLA2. The isoforms Cdr-12 and Cdr-13 had a sequence of amino acids of 122 amino acid residues, being Cdr-12: SLLQFNKMIK FETRKNAIPF YAFYGCYCGW GGQGRPKDAT DRCCIVHDCC YGKLAKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGYMIY PDSRCREPSE TC and pI value 8.37 and Cdr-13: SLVQFEKMIK EETGKNAVPF YAFYGCYCGW GGRGRPKDAT DRCCIVHDCC YEKLVKCNTK WDFYRYSLRS GYFQCGKGTW CEQQICECDR VAAECLRRSL STYRYGKMIY PDSRCREPSE TC with a pI value of 8.13 This sequence shows high identity values when compared to other D49 PLA2s isolated from venoms of crotalics snakes. Skeletal muscle preparations from the young chicken have been previously used in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. In mice, the PLA2 isoforms Cdr-12 and Cdr-13 induced myonecrosis and edema, upon intramuscular or subcutaneous injections, respectively. In vitro, Cdr-12 and Cdr-13 isoforms of PLA2, caused a potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and produced cytotoxicity in murine C2C12 skeletal muscle myotubes and lack cytolytic activity upon myoblasts in vitro. Thus, the combined structural and functional information obtained identify Cdr-12 and Cdr-13 isoforms as members of the PLA2 family, which presents the typical bioactivities described for such proteins.


Subject(s)
Crotalid Venoms/enzymology , Phospholipases A/chemistry , Phospholipases A/toxicity , Amino Acid Sequence , Animals , Cells, Cultured , Chemical Fractionation , Chickens , Crotalid Venoms/chemistry , Crotalid Venoms/isolation & purification , Crotalid Venoms/toxicity , Crotalus , Diaphragm/drug effects , Isoelectric Point , Isoenzymes , Mice , Molecular Weight , Muscle, Skeletal/pathology , Myoblasts/drug effects , Myoblasts/metabolism , Necrosis/pathology , Phospholipases A/isolation & purification , Phospholipases A2 , Phrenic Nerve/drug effects , Sequence Alignment , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
14.
Article in English | MEDLINE | ID: mdl-16997639

ABSTRACT

A basic toxin from Russell's viper venom of 7.2 kDa (RVV-7) has been purified to homogeneity after partial unfolding by 4 M urea followed by filtration through Centricon-30 membrane. Its N-terminal sequence showed strong homology with snake venom cytotoxins. Cytotoxic activity of RVV-7 has been demonstrated with B16F10 melanoma cells. PLA2 activity was observed in cytotoxin (CX3) from Naja kauthia bearing sequence homology with RVV-7. Phospholipase A2 and trypsin inhibitory activities were also observed with RVV-7. Chemical modification and inhibition studies suggested independent functional sites for these activities. A qualitative assessment of tumor growth inhibition by RVV-7 has been made.


Subject(s)
Cytotoxins/isolation & purification , Phospholipases A/isolation & purification , Viper Venoms/enzymology , Amino Acid Sequence , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Cytotoxins/chemistry , Cytotoxins/pharmacology , Male , Melanoma , Mice , Molecular Sequence Data , Molecular Weight , Neoplasm Transplantation , Phospholipases A/metabolism , Phospholipases A2 , Daboia , Sequence Alignment , Spectrometry, Mass, Electrospray Ionization , Trypsin Inhibitors/metabolism
15.
Biochimie ; 89(3): 319-28, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17110015

ABSTRACT

Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.


Subject(s)
Bothrops/metabolism , Phospholipases A/metabolism , Snake Venoms/enzymology , Amino Acid Sequence , Animals , Anticoagulants/pharmacology , Blood Coagulation/drug effects , Calcium/metabolism , Carcinoma, Ehrlich Tumor/chemically induced , Carcinoma, Ehrlich Tumor/enzymology , Carcinoma, Ehrlich Tumor/pathology , Cell Survival/drug effects , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Enzyme-Linked Immunosorbent Assay , Hemoglobins/metabolism , Humans , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , K562 Cells , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Molecular Weight , Phospholipases A/chemistry , Phospholipases A/isolation & purification , Platelet Aggregation/drug effects , Sequence Analysis, Protein , Sequence Homology, Amino Acid , Snake Venoms/pharmacology
16.
Protein J ; 25(7-8): 492-502, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17123155

ABSTRACT

Cr 5 PLA(2) homologous (K49) was isolated from Calloselasma rhodostoma venom in only one chromatographic step in reverse phase HPLC (RP-HPLC) (on mu-Bondapack C-18). A molecular mass of 13.965 Da was determined by MALDI-TOF mass spectrometry. The amino acid composition showed that Cr 5 had a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical residues of a basic PLA(2). The complete amino acid sequence of Cr 5 PLA(2) contains 120 residues, resulting in a calculated pI value of 5.55. This sequence shows high identity values when compared to other K49 PLA(2)s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA(2)s. The sequence found was SLVELGKMIL QETGKNPAKS YGAYGCNCGV LGRHKPKDAT DRCCFVHKCC YKKLTGCDPK KDRYSYSWKD KTIVCGENNP CLKEMCECDK AVAICLRENL DTYNKKYRYL KPFCKKADDC. In mice, Cr 5 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr 5 was 0.070 mg/kg of the animal weight, by intracerebroventricular (i.c.v.) route. In vitro, the toxin caused rapid cytolytic effect upon mouse skeletal muscle myoblasts in culture. The isolation of this PLA(2) and the combined structural and functional information obtained classify Cr 5 as a new member of the K49 PLA(2) family, since it presents typical features from such proteins.


Subject(s)
Crotalid Venoms/chemistry , Phospholipases A/isolation & purification , Viperidae/metabolism , Amino Acid Sequence , Animals , Cells, Cultured , Edema/chemically induced , Mice , Molecular Sequence Data , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/drug effects , Myoblasts/cytology , Myoblasts/drug effects , Phospholipases A/chemistry , Phospholipases A/toxicity , Phospholipases A2 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
17.
J Lipid Res ; 47(12): 2656-67, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17005997

ABSTRACT

The main triglyceride-lipase (TG-lipase) from the fat body of Manduca sexta has been identified as the homolog of Drosophila melanogaster CG8552. This protein is conserved among insects and also shares significant sequence similarity with vertebrate phospholipases (PLs) from the phosphatidic acid preferring-phospholipase A1 (PA-PLA(1)) family. It is shown here that the TG-lipase is also a PL. TG-lipase and PL activities copurify and are inhibited by, or resistant to, the same lipase inhibitors, indicating that both activities are catalyzed by the same enzyme and active site. The PL activity of TG-lipase corresponded to PL type A(1). The concentration dependence of lipase activity with TG and PL micellar substrates showed saturation kinetics, with apparent K(m) values of 152 +/- 11 and 7.8 +/- 1.1 muM, respectively. TG-lipase was able to hydrolyze the major phospholipid components of the lipid droplets, phosphatidylcholine and phosphatidylethanolamine. The enzyme hydrolyzes 77 molecules of TG for every molecule of PL contained in the lipid droplets. It was observed that the activation of lipolysis in vivo is accompanied by activation of the hydrolysis of phospholipids of the lipid droplets. These results suggest that the PL activity of the insect TG-lipase could be required to allow access of the lipase to TG molecules contained in the core of the lipid droplets.


Subject(s)
Fat Body/enzymology , Lipase/metabolism , Manduca/enzymology , Phospholipases A/metabolism , Amino Acid Sequence , Animals , Base Sequence , DNA, Complementary/genetics , Enzyme Inhibitors/pharmacology , Insect Hormones/metabolism , Insect Proteins/genetics , Insect Proteins/isolation & purification , Insect Proteins/metabolism , Lipase/genetics , Lipase/isolation & purification , Lipolysis , Manduca/genetics , Models, Biological , Molecular Sequence Data , Oligopeptides/metabolism , Phospholipases A/genetics , Phospholipases A/isolation & purification , Pyrrolidonecarboxylic Acid/analogs & derivatives , Pyrrolidonecarboxylic Acid/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Species Specificity , Substrate Specificity , Tandem Mass Spectrometry
18.
Article in English | MEDLINE | ID: mdl-16880551

ABSTRACT

For the first time, a complete X-ray diffraction data set has been collected from a myotoxic Asp49-phospholipase A2 (Asp49-PLA2) with low catalytic activity (BthTX-II from Bothrops jararacussu venom) and a molecular-replacement solution has been obtained with a dimer in the asymmetric unit. The quaternary structure of BthTX-II resembles the myotoxin Asp49-PLA2 PrTX-III (piratoxin III from B. pirajai venom) and all non-catalytic and myotoxic dimeric Lys49-PLA2s. In contrast, the oligomeric structure of BthTX-II is different from the highly catalytic and non-myotoxic BthA-I (acidic PLA2 from B. jararacussu). Thus, comparison between these structures should add insight into the catalytic and myotoxic activities of bothropic PLA2s.


Subject(s)
Asparagine , Bothrops , Crotalid Venoms/toxicity , Phospholipases A/toxicity , Animals , Catalysis , Crotalid Venoms/chemistry , Crotalid Venoms/metabolism , Crystallography, X-Ray , Kinetics , Phospholipases A/chemistry , Phospholipases A/isolation & purification , Phospholipases A/metabolism , Phospholipases A2 , Protein Conformation
19.
Protein J ; 25(2): 147-55, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16862457

ABSTRACT

In this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA(2) D49 purified from the Bothrops jararacussu venom. The isoforms 6-1 and 6-2 had a sequence of amino acids of 121 amino acid residues 6-1: DLFEWGQMIL KETGKNPFPY YGAYGCYCGW GGRGKPKDKD TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C and pI value 7.83 and 6-2: DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C with a pI value of 7.99. Skeletal muscle preparations from the young chicken have been used previously in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. Both isoforms have produced neuromuscular blockade in young chicken biventer cervicis nerve-muscle preparations in presence or absence of crotapotin crotalic (F3 and F4) indicating that catalytic activity was not essential for neuromuscular action in this preparation.


Subject(s)
Bothrops , Crotalid Venoms/enzymology , Neuromuscular Junction/drug effects , Phospholipases A/chemistry , Phospholipases A/toxicity , Amino Acid Sequence , Animals , Chickens , Crotalid Venoms/toxicity , Crotoxin/pharmacology , Isoenzymes/chemistry , Isoenzymes/isolation & purification , Isoenzymes/metabolism , Isoenzymes/toxicity , Molecular Sequence Data , Phospholipases A/isolation & purification , Phospholipases A/metabolism , Phospholipases A2 , Sequence Homology, Amino Acid
20.
Protein Expr Purif ; 50(1): 82-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-16809051

ABSTRACT

A secreted phospholipase A(2) (PLA(2)) from Streptomyces violaceoruber A-2688, previously identified by us, is the first PLA(2) identified in prokaryotes. Genome sequence data of Streptomyces coelicolor A3(2) indicates that the bacterium carries two genes encoding hypothetical PLA(2)s, which exhibit 100 and 78% identity, respectively, to the S. violaceoruber PLA(2). In this study, we named the former and latter proteins as the first and second PLA(2)s, respectively. When the second PLA(2) was expressed in Escherichia coli cells, it formed an inclusion body. The present study demonstrates a method to purify it to homogeneity without the disappearance of the enzymatic activity: the inclusion body was washed with sodium deoxycholate and dissolved in the presence of 2 M urea at pH 12, then refolded by the dilution method. The refolding of enzyme was confirmed by the circular dichroism spectrum. The second PLA(2) purified to homogeneity had the same specific activity as that of the S. violaceoruber PLA(2) and the yield was approximately 6.8 mg/L culture. The second PLA(2) exhibits similar enzymatic properties to the S. violaceoruber PLA(2), except that the former enzyme does not utilize phophatidic acid as a substrate. The surface electrostatic potential of the S. coelicolor PLA(2) model, which is created by the computer-homology modeling, suggests that the positively charged surface of the enzyme does not affect the substrate specificity.


Subject(s)
Inclusion Bodies/chemistry , Phospholipases A/chemistry , Phospholipases A/isolation & purification , Streptomyces coelicolor/enzymology , Amino Acid Sequence , Circular Dichroism , Cloning, Molecular , Crystallography, X-Ray , Enzyme Activation , Escherichia coli/genetics , Gene Expression Regulation, Enzymologic/genetics , Hydrogen-Ion Concentration , Inclusion Bodies/genetics , Kinetics , Models, Molecular , Molecular Sequence Data , Phospholipases A/genetics , Phospholipases A2 , Protein Folding , Sensitivity and Specificity , Sequence Alignment
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