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1.
Two phospholipases A2 (PLA2) fromNaja naja atra andNaja nigricollis snake venoms were subjected to tyrosine modification withp-nitrobenzenesulfonyl fluoride (NBSF) atpH 8.0. Three major NBS derivatives from each PLA2 were separated by high-performance liquid chromatography. The results of amino acid analysis showed that only two Tyr residues out of nine were modified, and the modified residues were identified to be Tyr-3 and Tyr-63 (or Tyr-62) in the sequence. Spectrophotometric titration indicated that the phenolic group of Tyr-3 and Tyr-63 (or Tyr-62) had apK of 10.1 and 11.0, respectively. The reactivity of Tyr-3 toward NBSF was not affected in the presence or absence of Ca
2+; however, the reactivity of Tyr-63 (or Tyr-62) toward NBSF was greatly enhanced by Ca2+. Modification of Tyr-63 (or Tyr-62) resulted in a marked decrease in both lethality and enzymatic activity. Conversely, modification of Tyr-3 inN. naja atra PLA2 could cause more than a sixfold increase in lethal potency, in sharp contrast to the loss of enzymatic activity.Tyrosine-63-modifiedN. naja atra PLA2 exhibited the same Ca2+-induced difference spectra as that of native PLA2, indicating that the Ca2+-binding ability of Tyr-63-modifiedN. naja atra PLA2 was not impaired. However, Tyr-3-modified PLA2 and all Tyr-modifiedN. nigricollis CMS-9 were not perturbed by Ca2+, revealing that the Ca2+-binding ability have been lost after tyrosine modification. These results suggest that Tyr-62 inN. nigricollis CMS-9 and Tyr-3 in both enzymes are involved in Ca2+ binding. AtpH 8.0, both native PLA2 enzymes enhance the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, while all of the Tyr-modified derivatives did not enhance the emission intensity at all either in the presence or absence of Ca2+, suggesting that the hydrophobic pocket that interacts with ANS might be the substrate binding site, in which Tyr-3 and Tyr-63 (or Tyr-62) are involved. 相似文献
2.
TANG Liang LIN ZhengjiongZHOU Yuancong 《中国科学:生命科学英文版》1997,40(5):481-487
Agkistrodotoxin, a neutral phospholipase A2 with high presynaptic neurotoxicity from the venom of Agkistrodon halys Pallas, has been crystallized by hanging drop vapor diffusion method. The crystal belongs to P21 space group with the cell dimensions a = 10.836 nm, b=8.486nm, c = 7.082nm, β=109.87$ showing C2 pseu-do-symmetry. Diffraction data to 0. 26 nm resolution have been collected on a Siemens X-200B area detector. C2 pseu-do-symmetry suggests that there exists a non-crystallographic two-fold axis parallel to crystallographic b axis. Self-rotation function calculation with different integrated radius and resolution ranges using the program POLARRFN yields four stable high peaks corresponding to three more non-crystallographic two-fold axis and one special non-crystal-lographic symmetry. The molecules in the asymmetric unit are suggested to be arranged in a manner of "dimer of dimers" by inference. 相似文献
3.
Three phospholipases A2 purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A2 activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A2, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca2+, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca2+ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca2+. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca2+ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A2 enzyme with the substrate, suggests that the Trp residues in phospholipase A2 enzymes and presynaptic toxins are involved in substrate binding. 相似文献
4.
Long-sen Chang Kou-wha Kuo Shinne-ren Lin Chun-chang Chang 《Journal of Protein Chemistry》1994,13(7):641-648
Phospholipase A2 (PLA2) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA2 and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA2 and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA2. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA2 and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA2 activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca2+ binding and antigenicity of Lys-6-modified PLA2 were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA2 to 32% of that of PLA2. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA2 activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA2 is almost the same as that of native PLA2. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA2, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA2. 相似文献
5.
Jen-Yirng Chiou Long-Sen Chang Lyn-Nou Chen Chun-Chang Chang 《Journal of Protein Chemistry》1995,14(6):451-456
A novel phospholipase A2, designated as Oh-DE-2, was isolated from the venom ofOphiophagus hannah (king cobra) by successive chromatography on SP-Sephadex C-25, DE-52, and Q-Sepharose columns. Oh-DE-2 with pI 5.1 showed an apparent molecular weight of 14 kD as revealed by SDS-PAGE and gel filtration. The amino acid sequence was homologous with those of PLA2s from Elapidae venoms. Oh-DE-2 was effectively inactivated byp-bromophenacyl bromide, indicating that the conserved His-48 is essential for its enzymatic activity. However, modification of the conserved Trp-19 did not cause a precipitous drop in the enzymatic activity of Oh-DE-2 as observed with PLA2s fromNaja naja atra andBungarus multicinctus venoms. A quenching study showed that the microenvironment of Trp in Oh-DE-2 was inaccessible to acrylamide, iodide, or cesium, a finding which was different from those observed with PLA2s fromN. naja atra andB. multicinctus venoms. These results might suggest that, unlike other PLA2 enzymes, Trp-19 in Oh-DE-2 is not directly involved in its enzymatic mechanisms. 相似文献
6.
Kohshi Kinkawa Shin Watanabe Kyozo Hayashi Seiji Inoue 《Biochemical and biophysical research communications》2010,395(3):377-381
Venomous snakes such as Gloydius brevicaudus have three distinct types of phospholipase A2 inhibitors (PLIα, PLIβ, and PLIγ) in their blood so as to protect themselves from their own venom phospholipases A2 (PLA2s). Expressions of these PLIs in G. brevicaudus liver were found to be enhanced by the intramuscular injection of its own venom. The enhancement of gene expressions of PLIα and PLIβ in the liver was also found to be induced by acidic PLA2 contained in this venom. Furthermore, these effects of acidic PLA2 on gene expression of PLIs were shown to be unrelated to its enzymatic activity. These results suggest that these venomous snakes have developed the self-protective system against their own venom, by which the venom components up-regulate the expression of anti-venom proteins in their liver. 相似文献
7.
Ji-Fu Wei Xiao-long Wei Qiu-Yu Chen Tian Huang Li-Ya Qiao Wan-Yu Wang Yu-Liang Xiong Shao-Heng He 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup. 相似文献
8.
Human non-pancreatic secretory phospholipase A2 (hnpsPLA2) is a group IIA phospholipase A2 which plays an important role in the innate immune response. This enzyme was found to exhibit bactericidal activity toward Gram-positive bacteria, but not Gram-negative ones. Though native hnpsPLA2 is active over a broad pH range, it is only highly active at alkaline conditions with the optimum activity pH of about 8.5. In order to make it highly active at neutral pH, we have obtained two hnpsPLA2 mutants, Glu89Lys and Arg100Glu that work better at neutral pH in a previous study. In the present study, we tested the bactericidal effects of the native hnpsPLA2 and the two mutants. Both native hnpsPLA2 and the two mutants exhibit bactericidal activity toward Gram-positive bacteria. Furthermore, they can also kill Escherichia coli, a Gram-negative bacterium. The two mutants showed better bactericidal activity for E. coli at neutral pH than the native enzyme, which is consistent with the enzyme activities. As hnpsPLA2 is highly stable and biocompatible, it may provide a promising therapy for bacteria infection treatment or other bactericidal applications. 相似文献
9.
Sequence homologues of the bacterium Streptomyces violaceoruber and sea anemone Nematostella vectensis PLA2 pfam09056 members were identified in several bacteria, fungi and metazoans illustrating the evolution of this PLA2 sub-family. Comparison of their molecular structures revealed that bacteria and fungi members are part of the GXIV of PLA2s while metazoan representatives are similar with GIX PLA2 of the marine snail Conus magus. Members of GXIV and GIX PLA2s show modest overall sequence similarity (21–35%) but considerable motif conservation within the putative Ca2+-binding, catalytic sites and cysteine residue positions which are essential for enzyme function. GXIV PLA2s of bacteria and fungi typically contain four cysteine residues composing two intramolecular disulphide bonds. GIX PLA2 homologues were identified in cnidarians and molluscs and in a single tunicate but appear to be absent from other metazoan genomes. The mature GIX PLA2 deduced peptides contain up to ten cysteine residues capable of forming five putative disulphide bonds. Three disulphide bonds were identified in GIX PLA2s, two of which correspond to those localized in GXIV PLA2s. Phylogenetic analysis demonstrates that metazoan GIX PLA2s cluster separate from the bacterial and fungal GXIV PLA2s and both pfam09056 members form a group separate from the prokaryote and eukaryote GXIIA PLA2 pfam06951. Duplicate PLA2 pfam09056 genes were identified in the genomes of sea anemone N. vectensis and oyster Crassostrea gigas suggest that members of this family evolved via species-specific duplication events. These observations indicate that the newly identified metazoan pfam09056 members may be classified as GIX PLA2s and support the idea of the common evolutionary origin of GXIV and GIX PLA2 pfam09056 members, which emerged early in bacteria and were maintained in the genomes of fungi and selected extant metazoan taxa. 相似文献
10.
Fluorescence measurements of the homologous proteins, notexin and PLA2 enzymes fromNaja naja atra, Naja nigricollis, and Hemachatus haemachatus venoms, showed that the wavelength of maximum emission and the quantum yield of their intrinsic fluorescence emission spectra were different. To verify the factors which affected their fluorescence characteristics, the dynamics of tryptophan residues in those homologous proteins were studied by quenching with acrylamide, iodide, and cesium. The degrees of exposure of tryptophanyl groups in notexin and PLA2 enzymes assessed by acrylamide quenching were found to be the major factor that determined their fluorescence characteristics. However, the positively charged groups surrounding tryptophan residues of PLA2 enzymes fromN. naja atra andN. nigricollis venoms might affect the quantum yield of their fluorophores. Tryptophan residues of notexin were in an environment with less fluctuation, which did not allow free diffusion of ionic quencher. This might render its typtophan residues to fluoresce at a shorter wavelength. These results suggested that the structural determinants affecting the intrinsic fluorescence emission of homologous proteins can be easily assessed by quenching studies. 相似文献
11.
Phospholipase A2 (PLA2) enzymes catalyze the hydrolysis of the sn-2 position of glycerophospholipids to produce free fatty acids and lysophospholipids. More than one third of the mammalian PLA2 enzymes belong to the secreted PLA2 (sPLA2) family, which consists of low molecular mass, Ca2+-requiring enzymes with a His–Asp catalytic dyad. Individual sPLA2 enzymes exhibit unique tissue and cellular localizations and specific enzymatic properties, suggesting their distinct biological roles. The past decade has met a new era of the sPLA2 research field toward deciphering their in vivo functions by developing several specific tools and methods. These include i) the production of transgenic and knockout mouse lines for several sPLA2s, ii) the development of specific analytical tools including the production of large amounts of recombinant sPLA2 proteins, and iii) applying mass spectrometry lipidomics to unveil their specific enzymatic properties occurring in vivo. It is now obvious that individual sPLA2s are involved in diverse biological events through lipid mediator-dependent and -independent processes, act redundantly or non-redundantly in the context of physiology and pathophysiology, and may represent potential drug targets or novel bioactive molecules in certain situations. In this review, we will highlight the newest understanding of the biological roles of sPLA2s in the past few years. 相似文献
12.
Julián Fernández José María Gutiérrez Yamileth Angulo Libia Sanz Paula Juárez Juan J. Calvete Bruno Lomonte 《Biochimie》2010
Phospholipases A2 (PLA2) are major components of snake venoms, exerting a variety of relevant toxic actions such as neurotoxicity and myotoxicity, among others. Since the majority of toxic PLA2s are basic proteins, acidic isoforms and their possible roles in venoms are less understood. In this study, an acidic enzyme (BaspPLA2-II) was isolated from the venom of Bothrops asper (Pacific region of Costa Rica) and characterized. BaspPLA2-II is monomeric, with a mass of 14,212 ± 6 Da and a pI of 4.9. Its complete sequence of 124 amino acids was deduced through cDNA and protein sequencing, showing that it belongs to the Asp49 group of catalytically active enzymes. In vivo and in vitro assays demonstrated that BaspPLA2-II, in contrast to the basic Asp49 counterparts present in the same venom, lacks myotoxic, cytotoxic, and anticoagulant activities. BaspPLA2-II also differed from other acidic PLA2s described in Bothrops spp. venoms, as it did not show hypotensive and anti-platelet aggregation activities. Furthermore, this enzyme was not lethal to mice at intravenous doses up to 100 μg (5.9 μg/g), indicating its lack of neurotoxic activity. The only toxic effect recorded in vivo was a moderate induction of local edema. Therefore, the toxicological characteristics of BaspPLA2-II suggest that it does not play a key role in the pathophysiology of envenomings by B. asper, and that its purpose might be restricted to digestive functions. Immunochemical analyses using antibodies raised against BaspPLA2-II revealed that acidic and basic PLA2s form two different antigenic groups in B. asper venom. 相似文献
13.
To determine whether the ability to induce neurite outgrowth in rat pheochromocytoma cell line PC12 is characteristic of phospholipases of different types, we have studied the influence of phospholipase A(2) (PLA2) from cobra Naja kaouthia venom and two PLA2s from viper Vipera nikolskii venom on PC12 cells. Phospholipases from the viper venom are heterodimers in which only one of the subunits is enzymatically active, while PLA2 from the cobra venom is a monomer. It was found that all three PLA2s induce neurite outgrowth in PC12. The PLA2 from cobra venom exhibits this effect at higher concentrations as compared to the viper enzymes. We have not observed such an activity for isolated subunits of viper PLA2s, since the enzymatically active subunits have very high cytotoxicity, while the other subunits are not active at all. However, co-incubation of active and inactive subunits before addition to the cells leads to a marked decrease in cytotoxicity and to restoration of the neurite-inducing activity. It has also been shown that all enzymatically active PLA2s are cytotoxic, the PLA2 from cobra venom being the least active. Thus, for the first time we have shown that PLA2s from snake venoms can induce neurite outgrowth in PC12 cells. 相似文献
14.
Investigations have been carried out on the influence of the phospholipid composition and the physicochemical properties of rat liver plasma membranes on the endogenous activity of membrane-bound phospholipase A2. The membrane phospholipid composition was modified by the incorporation of different phospholipids in the lipid bilayer by the aid of lipid transfer proteins. The results indicate that the endogenous activity of phospholipase A2 in liver plasma membranes depends upon membrane fluidity and not upon the presence of a specific phospholipid in the enzyme's microenvironment. 相似文献
15.
16.
Kieran F. Scott Mila Sajinovic Juliane Hein Sheri Nixdorf Peter Galettis Winston Liauw Paul de Souza Qihan Dong Garry G. Graham Pamela J. Russell 《Biochimie》2010
Phospholipase A2 (PLA2) enzymes (EC3.1.4.4) regulate the release of biologically active fatty acids and lysophospholipids from membrane phospholipid pools. These lipids are also substrates for intracellular biochemical pathways that generate potent autocrine and paracrine lipid mediators such as the eicosanoids and platelet activating factor. These factors, in turn, regulate cell proliferation, survival, differentiation, motility, tissue vascularisation, and immune surveillance in virtually all tissues, functions that are subverted by cancer cells for tumour growth and metastasis. Thus the relevance of PLA2-dependent pathways to the genesis and progression of cancer has been of interest since their discovery and with recent technological advances, their role in tumourigenesis has become more tractable experimentally. Limited human genetic studies have not yet identified PLA2 enzymes as classical mutated oncogenes or tumour suppressor genes. However, there is strong evidence that of the 22 identified human PLA2 enzymes, ten of which have been studied in cancer to date, most are aberrantly expressed in a proportion of tumours derived from diverse organs. Correlative and functional studies implicate the expression of some secreted enzymes (sPLA2s), particularly the best studied enzyme Group IIA sPLA2 in either tumour promotion or inhibition, depending on the organ involved and the biochemical microenvironment of tumours. As in immune-mediated inflammatory pathologies, genetic deletion studies in mice, supported by limited studies with human cells and tissues, have identified an important role for Group IVA PLA2 in regulating certain cancers. Pharmacological intervention studies in prostate cancer suggest that hGIIA-dependent tumour growth is dependent on indirect regulation of Group IVA PLA2. Group VI calcium-independent PLA2 enzymes have also been recently implicated in tumourigenesis with in vitro studies suggesting multiple possible roles for these enzymes. Though apparently complex, further characterization of the regulatory relationships amongst PLA2 enzymes, lipid mediator biosynthetic enzymes and the lipid mediators they produce during tumour progression is required to define the biochemical context in which the enzymes modulate cancer growth and development. 相似文献
17.
Among the emerging phospholipase A2 (PLA2) superfamily, the secreted PLA2 (sPLA2) family consists of low-molecular-mass, Ca2+-requiring extracellular enzymes with a His-Asp catalytic dyad. To date, more than 10 sPLA2 enzymes have been identified in mammals. Individual sPLA2s exhibit unique tissue and cellular localizations and enzymatic properties, suggesting their distinct pathophysiological roles. Despite numerous enzymatic and cell biological studies on this enzyme family in the past two decades, their precise in vivo functions still remain largely obscure. Recent studies using transgenic and knockout mice for several sPLA2 enzymes, in combination with lipidomics approaches, have opened new insights into their distinct contributions to various biological events such as food digestion, host defense, inflammation, asthma and atherosclerosis. In this article, we overview the latest understanding of the pathophysiological functions of individual sPLA2 isoforms fueled by studies employing transgenic and knockout mice for several sPLA2s. 相似文献
18.
A phospholipase A2 was isolated from the snake venom of Chinese Agkistrodon blomhoffii Ussurensis by column chromatography using DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration chromatography and Mono Q ion-exchange chromatography, and designated as Akbu-PLA2. It showed an average molecular mass of 13,980 ± 3 amu determined by MALDI TOF mass spectrometry. Protein identification results from HPLC-nESI-MS/MS analysis indicated that the Akbu-PLA2 was a new snake venom acidic PLA2. Seven peptides were sequenced from Akbu-PLA2 by HPLC-nESI-MS/MS analysis. Sequencing alignment indicated that Akbu-PLA2 shared homolog peptides of phospholipases A2 from the venoms of Gloydius ussurensis, Gloydius halys, Gloydius halys (halys viper), Deinagkistrodon acutus and Agkistrodon halys Pallas. Akbu-PLA2 has an optimum hydrolytic activity temperature of ∼45 °C. The intrinsic fluorescences of Tyr and Trp residues of Akbu-PLA2 showed emission wavelengths red-shifted by 13.6 and 1.6 nm from those of free Tyr and Trp, respectively. Akbu-PLA2 was shown to contain one Ca2+ per monomer by ICP-AES measurement. The Ca2+ ion was found to be critical for both the hydrolytic activity and the structure of Akbu-PLA2. Ca2+ increased the emission fluorescence intensity and the hydrophobicity of the environment of Akbu-PLA2. The hydrolytic activity of Akbu-PLA2 was accelerated due to the addition of Ca2+ ion by enhancing the substrate binding. However, a protein component with the molecular weight two-fold relative to that of Akbu-PLA2 was found to be difficult to eliminate for the purification of Akbu-PLA2. HPLC-nESI-MS/MS detected the same peptides from it as from Abku-PLA2, which indicated that it should be a homodimer of Akbu-PLA2. A proteomic approach, 2D SDS-PAGE coupled to HPLC-nESI-MS/MS, supported the co-existence of the Akbu-PLA2 monomer and dimer in the crude snake venom. Results from the combination of phosphoprotein and glycoprotein specific stains combined with the HPLC-nESI-MS/MS method indicated that both the Akbu-PLA2 monomer and dimer were both phosphorylated and glycosylated. The addition of exogenous Ca2+ ion was found to be able to promote the dimer formation of Akbu-PLA2. We conclude that a novel PLA2 was successfully obtained. The systemically biochemical, proteomic, structural and functional characterization results from Akbu-PLA2 reveal new threads and provide valuable inputs for the study of snake venom phospholipases A2. 相似文献
19.
Structural and functional characterization of myotoxin, Cr-IV 1, a phospholipase A2 D49 from the venom of the snake Calloselasma rhodostoma 总被引:1,自引:0,他引:1
V.L. Bonfim L.A. Ponce-Soto D. Martins de Souza G.H.M.F. Souza P.A. Baldasso M.N. Eberlin S. Marangoni 《Biologicals》2008,36(3):168-176
A new D49 PLA(2) was purified from the venom of Calloselasma rhodostoma after two chromatographic steps. Molecular exclusion chromatography was done through a Protein-Pack 300 SW column (0.78 cm x 30 cm), eluting with 0.25 M ammonium bicarbonate, pH 7.9, at a flow rate of 0.3 ml/min. Reverse-phase HPLC was then performed on mu-Bondapack C-18. The sample was determined to have a molecular mass of 13,870.94 Da MALDI-TOF by mass spectrometry, and the amino acid composition showed that Cr-IV 1 presented a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). Cr-IV 1 presented a sequence of 122 amino acid residues: DLWEFGQMILKETGSLPFPY YTTYGCYCGV GGRGGKPKDA TDRCCFVHDC CYGKLTGCPK TNDRYSYSRL DYTIVCGEGG PCKQICECDK AAAVCFRENL RTYNKKYRYHLKPFCKEPAE TC and a calculated pI value of 8.0. Cr-IV 1 had PLA(2) activity in the presence of a synthetic chromogenic substrate (4-nitro-3-(octanoyloxy)benzoic acid) and showed a rapid cytolytic effect on mouse skeletal muscle myoblasts and myotubes in culture. In mice, Cr-IV 1 induced myonecrosis and edema upon intramuscular and intravenous injections, respectively. The LD(50) of Cr-IV 1 was determined to be 0.07 mg/k body weight by intracerebroventricular (i.c.v.) injection. The combination of structural and functional information obtained herein classifies Cr-IV 1 as a new member of the D49 PLA(2) family, as it presents the typical behavior of a phospholipase A(2) from this family. 相似文献
20.
Luis Alberto Ponce-Soto Bruno Lomonte José María Gutiérrez Lea Rodrigues-Simioni José Camillo Novello Sergio Marangoni 《Biochimica et Biophysica Acta (BBA)/General Subjects》2007
BaTX PLA2, a K49 phospholipase A2 homologue was purified from Bothrops alternatus venom after two chromatographic steps, molecular exclusion on Superdex 75 and reverse phase HPLC on μ-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 PLA2. The complete amino acid sequence of BaTX PLA2 contains 121 residues, resulting in a calculated pI value of 8.63. This sequence shows high identity values when compared to other K49 PLA2s isolated from the venoms of viperid snakes. Lower identity is observed in comparison to D49 PLA2s. 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 LD50 of BaTX was 7 μg/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 μM: 40 ± 0.4 min and 0.07 μM: 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 PLA2 family, which presents the typical bioactivities described for such proteins. 相似文献