共查询到20条相似文献,搜索用时 15 毫秒
1.
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and
. Effect of a phospholipase A 2 with cardiotoxin-like properties, from Bungarus fasciatus snake venom, on calcium-modulated potassium currents. Toxicon 27, 1339–1349, 1989.—The action of a 16,300mol. wt phospholipase A 2 with cardiotoxin-like properties from Bungarus fasciatus venom on membrane electrical properties of two human cell types was examined in vitro by using tight-seal whole-cell recording methods. Epithelial cells exhibited a voltage-and Ca 2+-activated K +current; the sensitivity for voltage activation of the K + current was enhanced by increasing free Ca 2+ in the recording pipette from 10 −8 M to 2 × 10 −6 M. In contrast, peripheral blood lymphocytes possessed voltage-activated K + currents that were inhibited by increasing intracellular Ca 2+. Exposure of either preparation to B.fasciatus toxin (0.2–5 × 10−6 M) for up to 30 min in the bath did not alter membrane leakage current, as judged by the maintenance of low pre-treatment values over the range of − 140mV to − 40mV. However, the sensitivity for voltage activation of the K+ current was enhanced in the epithelial cells even at the lowest concentrations tested. In contrast to the results with epithelial cells, toxin exposure inhibited the activation of voltage-activated K+ currents in human lymphocytes, suggesting a specific increase in intracellular Ca2+ levels in both cell types. The fluorescent probe indo-1/AM was used to monitor cytoplasmic Ca2+ levels. Exposure of either lymphocytes or epithelial cells to toxin (10−6 M) resulted in a transient increase in Ca2+. However, while the Ca2+ response to toxin was transient, K-channel modulation by the toxin appeared to be irreversible over the experimental time course. The longer-lasting modulation of Ca2+-regulated K+ channels may reflect an irreversible action of the B.fasciatus phospholipase A2 on a Ca2+-dependent regulatory process. 相似文献
2.
E.
and P.
. Inhibition of phosphorylation of rat synaptosomal proteins by snake venom phospholipase A 2 neurotoxins (β-bungarotoxin, notexin) and enzymes ( Naja naja atra, Naja nigricollis). Toxicon 28, 1423–1437, 1990.—Some snake venom neurotoxins, such as β-bungarotoxin (β-BuTX) and notexin, which inhibit the release of neurotransmitter at both peripheral and central presynaptic terminals possess phospholipase A 2 activity. In contrast, most snake venom phospholipase A 2 enzymes such as those isolated from Naja naja atra and Naja nigricollis are structurally homologous to these neutrotoxins but do not have any specific or potent presynaptic action although they have higher enzymatic activities than the neurotoxins. In order to investigate the mechanisms of presynaptic action of the snake venom neurotoxins, we studied their effects on phosphorylation of rat brain synaptosomal proteins. It is known that phosphorylation of synapsin I, a neuron specific and synaptic vesicle associated phosphoprotein, increases neurotransmitter release. Incubation of cerebral cortical synaptosomes with 32P- orthophosphate at 37°C for 30 min, caused significant phosphorylation of a wide mol. wt range of proteins including most markedly those proteins in the mol. wt range (81,000–86,000) of synapsin I. Both snake venom phospholipase A 2 neurotoxins and enzymes (5, 15 and 50 nM) inhibited phosphorylation in a Ca 2+-dependent manner with the following order of potencies: β- BuTX > N. n. atra phospholipase A2 ≥ notexin > N. nigricollis phospholipase A 2. Five nanomoles of β-BuTX, which has the lowest phospholipase A 2 activity, inhibited phosphorylation of a wide range of mol. wt proteins (51,000–188,000) by 42–58%. At the same concentration, N.n. atra phospholipase A 2 (which possesses the highest enzymatic activity), notexin and N. nigricollis phospholipase A 2 caused less inhibition than β-BuTX, ranging from 0–40% depending on the agent used. These results indicate that there is no correlation between their potencies in inhibiting phosphorylation and the levels of their phospholipase A 2 activities. An inhibitory activity on phosphorylation may be at least partially responsible for a presynaptically-induced block of neurotransmitter release. 相似文献
3.
A basic protein was isolated by CM-Sephadex C-25 chromatography from the venom of Bothrops neuwiedii from Argentina, and named B. neuwiedii myotoxin I. This protein exerted local myotoxic and edema-forming effects in mice, with potencies comparable to other myotoxins isolated from Bothrops spp. venoms. When injected by i.v. route at doses up to 4.7 mg/kg of body weight, the toxin was not lethal. In vitro, the toxin had no detectable phospholipase A 2 activity on egg yolk phospholipids. B. neuwiedii myotoxin I appeared as a homodimer in sodium dodecylsulphate–polyacrylamide gel electrophoresis, with a subunit molecular weight of 15 kD. Gel immunodiffusion revealed a pattern of partial antigenic identity between the newly isolated myotoxin and myotoxin II from Bothrops asper venom. The sequence of B. neuwiedii myotoxin I was determined for the first 40 amino acid residues, showing high homology to several class II phospholipase A 2 myotoxins of the Lys-49 family from crotalids. Altogether, results suggest that this toxin is a new member of the Lys-49 phospholipase A 2-homologues with myotoxic, cytolytic, and edema-inducing activities. 相似文献
4.
A phospholipase A 2 (EC 3.1.1.4) has been isolated and purified from the venom of Crotalus scutulatus salvini in 33% yield. The enzyme was homogeneous on polyacrylamide gel electrophoresis. The molecular weight of the enzyme was in the same range (30,000) whether determined by gel filtration or ultracentrifugation. On polyacrylamide gel electrophoresis, in the presence of sodium dodecyl sulphate and β-mercaptoethanol, the enzyme migrated into a single protein band with a mobility corresponding to about 14,000 molecular weight, indicating that the native enzyme was a dimer. Amino acid composition of the enzyme is reported. Two NH 2-amino acid terminals were found which indicates that the dissociable monomers of the native enzyme were non-identical. Antiserum against the purified enzyme completely inhibited the phospholipase A 2 activity and immunodiffusion of the antiserum against both purified phospholipase A 2 and crude venom of Crotalus scutulatus salvini gave single precipitin lines indicating a lack of contaminating antigens. 相似文献
5.
The immunological properties of phospholipase A 2 present in venoms from seven species of rattlesnakes (genus Crotalus) from different geographical distributions have been compared. Rabbit antiserum to purified PLA 2 of Crotalus scutulatus salvini venom was used, employing inhibition of enzymic activity and immunodiffusion techniques. The amount of antiserum required for 50% inhibition of the PLA 2 activity in the venoms varied from slightly more than that required for inhibition of the activity in the homologous venom to a large amount (effectively zero inhibition). A particularly close relationship in the antigenic makeup was observed between the phospholipases of C. s. salvini and C. atrox venoms as evidenced by inhibition of enzymic activity and immunodiffusion studies. No immunological cross reactivity was observed between the enzymes of the subspecies C. s. salvini and C. s. scutulatus. The results suggest extensive variations in the antigenic structure of the phospholipase molecules. The findings may also have some taxonomic significance. 相似文献
6.
The action of the neurotoxic complex and its components from the venom of the Bulgarian viper ( Vipera ammodytes ammodytes) on phosphatidylcholine was studied. The nontoxic acidic component partially inhibited the phospholipase A 2 activity of the strongly toxic basic component. The basic component, separated from the acidic, was unstable and in the course of 12–14 days lost its enzymatic activity. The Michaelis constant, Km = 1 × 10 −3M was the same for the free phospholipase A 2 and the neurotoxic complex. Temperature optimum was 23–26°C and pH optimum was 9.9–10. A concentration of 1–3 × 10 −3 M CaCl 2 was required for maximum enzyme activity. The influence of divalent cations on the initial velocity of the enzyme hydrolysis was studied. Although composed of a basic toxic phospholipase A 2 and a nontoxic acidic component the neurotoxic complex exhibited an insignificant enzyme activity. 相似文献
7.
A phospholipase A 2 inhibitor has been previously purified and cloned from the blood plasma of the South American rattlesnake, Crotalus durissus terrificus. This inhibitor, named CNF for Crotalus neutralizing factor, interacts with crotoxin, the main neurotoxin from C. d. terrificus venom, abolishing its phospholipase A 2 activity. Crotoxin is a heterodimer of an acidic subunit (CA) and a basic phospholipase A 2 (CB). CNF acts by forming a stable non-toxic complex with CB, replacing CA in the toxic CA–CB of crotoxin. In the present investigation, we have shown that CNF has a broader specificity. It is able to inhibit the PLA2 activity of the whole venom from the bushmaster snake (Lachesis muta muta), a species evolutionary related to Crotalus. Inhibition experiments have been carried out with four PLA2 active components isolated from L. m. muta venom, one basic and three acidic ones. CNF inhibition is not restricted to the basic PLA2, but extended to the three acidic forms as well. 相似文献
8.
Venoms from two related Australian ants, a jumper ant ( Myrmecia pilosula) and a bulldog ant ( Myrmecia pyriformis), were quantitatively analysed for the following enzymic activities: phospholipase A 2, phospholipase B, phospholipase C, hyaluronidase, esterase, acid phosphatase, alkaline phosphatase and phosphodiesterase. Both venoms contained phospholipase A 2, phospholipase B, hyaluronidase, acid phosphatase and alkaline phosphatase activities. Myrmecia pyriformis venom had significantly greater phospholipase B, acid phosphatase and alkaline phosphatase activities than Myrmecia pilosula venom. No detectable quantities of phospholipase C, esterase or phosphodiesterase activities were found in either venom. 相似文献
9.
Experiments with immobilized concanavalin A strongly suggest a glycoprotein nature of three honey-bee venom enzymes, phospholipase A 2, hyaluronidase and acid phosphatase. The electrophoretically and chromatographically detectable heterogeneity of phospholipase A 2 results from absence of carbohydrate in a subfraction. Mannose, fucose and N- acetylglucosamine, but not galactose nor f-tylgalactosamineare present in the con A-binding fraction of bee venom. It is therefore concluded that only N-glycosidically linked carbohydrate occurs in bee venom glycoproteins. 相似文献
10.
Studies on the inhibitory effects of a series of anti-snake-venom sera on the phospholipase A 2 activity of a number of snake venoms have been made using the suppression of phospholipase A 2 activity by the antiserum as an indicator of interaction between antibodies against phospholipase A 2 and snake venom phospholipase A 2. Specific antiserum against the venom of a particular species inhibited the phospholipase A 2 activity of the venoms of closely related species, but in most cases, failed to suppress phospholipase A 2 activity of venoms from other families. The amount of antiserum required for 50% inhibition of phospholipase A 2 activity varied with the species. The differences in the inhibitory properties of a particular antiserum on a heterologous phospholipase A 2 activity indicate variations in the antigenic sites of the enzyme molecule. 相似文献
11.
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. Effect of chemical modification with p-bromophenacyl bromide on the enzymatic and lethal properties of phospholipase A 2 from Bothrops alternatus (Vibora de la Cruz) venom. Toxicon 26, 1137–1144, 1988.—The effects on lethal potency and enzymatic activity were determined following alkylation, with p-bromophenacyl bromide, of the acidic toxic phospholipase A 2 from Bothrops alternatus. The modified B. alternatus enzyme, which lost its enzymatic activity, retained considerable toxicity. Histopathologic studies on mice have demonstrated features similar to those of the native enzyme. However, the distribution of the damage was different and the survival time was longer. It is concluded that the enzyme activity is not important for the lethal action of the enzyme although it influences the distribution of the damage and survival time. 相似文献
12.
Two monomeric neurotoxic phospholipases A 2 have been crystallized and their diffraction properties characterized. Crystals of caudoxin (from the venom of Bitis caudalis) and notexin (from the venom of Notechis scutatus scutatus) were grown at neutral pH, in the absence of calcium ion, and diffract to a resolution of 2.3 and 1.6 Å, respectively. 相似文献
13.
The localization and expression profiles of phospholipases A 2 (PLA 2s) in Trimeresurus flavoviridis venom gland were studied by means of in situ hybridization and immunohistochemical techniques. Venom gland cells are tightly arrayed in a single layer along the inlet-like lumens in which venom proteins are stored. mRNAs for PLA 2s were detected at the high level in cytoplasm. Using the immunohistochemical technique with polyclonal antiAsp-49-PLA 2 antibody, Asp-49-PLA 2 and, possibly, its isozymes were detected in intracellular granules and in venom lumens. The intracellular granules containing PLA 2 proteins appear to be transferred from the nucleus towards the outer membrane facing the lumen, and then to be secreted. 相似文献
14.
Bitis gabonica venom was separated on Sephadex G-50 and the major enzymes present in the venom localized by assaying the eluate against specific substrates. Phospholipase A was purified and its positional specificity towards phosphatidylcholine shown to be of the A 2 type. The amino acid composition of the purified phospholipase A is reported. An equilibrium is shown to exist between monomer and dimer which is favoured towards the dimer by addition of Ca 2+ and towards the monomer by EDTE. 相似文献
15.
R. M.
and H. J.
. Review article—Effects of snake venom proteins on blood platelets. Toxicon 28, 1387–1422, 1990.—Snake venoms are complex mixtures which contain pharmacologically active polypeptides and proteins. Several snake venom constituents interfere in platelet aggregation, an important cellular process in thrombosis and hemostasis. These components range in size from small molecular weight polypeptides to high molecular weight proteins. Some of the proteins are enzymes, such as phospholipase A 2, proteinases, nucleotidases, or
-amino acid oxidase, while others do not exhibit enzymatic activity. These components may initiate and/or inhibit platelet aggregation. Some venom factors induce platelet agglutination. This review deals with the physical characteristics of these venom factors, the mechanisms of their platelet effects, structure-function relationships, and their physiological significance. 相似文献
16.
The pharmacological modulation of edema-forming activity of Bothrops asper myotoxins II and III, Lys-49 and Asp-49 phospholipases A 2, respectively, was studied plethysmographically in the mouse foot pad model. Myotoxin III had phospholipase A 2 activity, whereas myotoxin II was devoid of enzymatic activity when tested on egg yolk phosphatidylcholine. Both toxins induced a dose-dependent edema of rapid onset. Chemical modification of myotoxin III with p-bromophenacyl bromide abrogated enzymatic activity and significantly reduced edema-forming activity, although a residual effect remained. Pretreatment of animals with diphenhydramine, dexamethasone, indomethacin and prazosin significantly reduced the effect of both myotoxins. It is concluded that (a) these myotoxins are important edema-forming components of B. asper venom, (b) enzymatic activity is not a strict requirement to exert this effect, although in the case of myotoxin III it contributes to its development, and (c) several inflammatory mediators participate in mouse foot pad edema induced by these myotoxins. 相似文献
17.
Two species of giant hornet phospholipase B (PLB), and β, were purified from the venom of Vespa mandarinia. The purification procedure was simplified by two steps of column chromatographies, Sephadex G-100 and SP-Sepharose. The molecular sizes of PLB and β were 29.5 and 26.0 kDa, respectively. The isoelectric point of and β enzymes were pH 10.6 and 10.7, respectively. The temperature optimum for egg yolk lecithin was a broad peak at 40–60°C for both enzymes. Amino acid compositions of both enzymes were high contents of aspartic acid, glycine, leucine, lysine and other aliphatic amino acids. Cystine was similar amounts to other species of phospholipases (PLs). The Km values of and β enzymes were 8.29 and 7.53 mg/ml for egg yolk lecithin, respectively. In the catalytic specificity for L--phosphatidylcholine-β-oleoil-γ-palmitoil, the Km values of enzyme for γ-palmitoil and β-oleoil residues were 0.528 and 1.392 mM, respectively. While the Km values of β enzyme for γ-palmitoil and β-oleoil residues were 7.91 and 2.68 mM, respectively. Both and β enzymes were inhibited strongly by cepharanthine. The lecithin hydrolysis of enzyme was competitively inhibited, but β enzyme was uncompetitive. Cepharanthine also inhibited noncompetitively PLA 2s of bovine pancreas, bee venom and Naja mossambica mossambica. 相似文献
18.
In addition to their catalytic activity, snake venom phospholipases A 2 (vPLA 2) present remarkable diversity in their biological effects. Sequence alignment analyses of functionally related PLA 2 are frequently used to predict the structural determinants of these effects, and the predictions are subsequently evaluated by site directed mutagenesis experiments and functional assays. In order to improve the predictive potential of computer-based analysis, a simple method for scanning amino acid variation analysis (SAVANA) has been developed and included in the analysis of the lysine 49 PLA 2 myotoxins (Lys49-PLA 2). The SAVANA analysis identified positions in the C-terminal loop region of the protein, which were not identified using previously available sequence analysis tools. Site directed mutagenesis experiments of bothropstoxin-I, a Lys49-PLA 2 isolated from the venom of Bothrops jararacussu, reveals that these residues are exactly those involved in the determination of myotoxic and membrane damaging activities. The SAVANA method has been used to analyse presynaptic neurotoxic and anti-coagulant vPLA 2s, and the predicted structural determinants of these activities are in excellent agreement with the available results of site directed mutagenesis experiments. The positions of residues involved in the myotoxic and neurotoxic determinants demonstrate significant overlap, suggesting that the multiple biological effects observed in many snake vPLA 2s are a consequence of superposed structural determinants on the protein surface. 相似文献
19.
Previous studies have shown that homologous phospholipases A 2 (PLA 2) (Pa-3, Pa-9C, Pa-10F and Pa-11) from the venom of the Australian king brown snake, Pseudechis australis, significantly reduce the resting membrane potentials and quantal contents of endplate potentials recorded from endplate regions of mouse triangularis sterni nerve-muscle preparations. It is not clear whether PLA 2 activity is essential for their neuromuscular activities. Therefore, pharmacological studies were carried out to determine whether neuromuscular activity of the toxins changed after treatment with the phospholipase A 2 inhibitors 7,7-dimethyl-eicosadienoic acid (DEDA) and manoalide. After incubation of the toxins with manoalide (120nM), or DEDA (50 μM), no PLA 2 activity against 1-stearoyl 2-[ 3H]arachidonoylglycerophosphocholine was detected. After incubation with manoalide and/or DEDA, the toxins did not depolarize muscle fibre membranes up to 60 min after administration. However, manoalide and DEDA had different influences on the inhibitory effect of these toxic enzymes on acetylcholine release from nerve terminals. Manoalide abolished the inhibitory effect of the toxins on evoked release of acetylcholine. In contrast, DEDA was not able to prevent the reduction of quantal content of endplate potentials induced by the toxins. This study provides evidence that the depolarizing action and the inhibitory effect on release of acetylcholine exerted by these toxic PLA 2 from king brown snake are independent phenomena. The evidence for this conclusion was that inhibition of enzymatic activity with an arachidonic acid analogue (DEDA) abolished the depolarizing effect of the toxins but not the effects on the quantal release of acetylcholine from mouse motor nerve terminals. The data suggest that the depolarizing effect of these toxins is probably due to the enzymatic activity. Since manoalide interacts with lysine residues of PLA 2 polypeptides, and, as shown here, manoalide prevented inhibition of neurotransmitter release, lysine residues may play an important role in the inhibitory activity of these toxins. 相似文献
20.
Acute pancreatitis (AP) is an inflammatory disease of the pancreas characterized by local inflammation and extrapancreatic effects such as lung injury. Secretory phospholipases A 2 (PLA 2s) have been implicated in triggering AP, but their exact role to evoke AP is largely unknown. Therefore, we have tested the ability of sPLA 2s to induce AP in rats, using venom sPLA 2s with residual or high enzymatic activity (bothropstoxin-II and Naja mocambique mocambique venom PLA 2, respectively), as well as sPLA 2 devoid of catalytic activity (piratoxin-I). The injection of Naja m. mocambique venom PLA 2, bothropstoxin-II or piratoxin-I (300 μg/kg each) into the common bile duct increased significantly the pancreatic plasma extravasation and myeloperoxidase activity. The lung myeloperoxidase and serum amylase were also increased for all groups, although the Naja mocambique mocambique venom PLA 2 induced higher lung myeloperoxidase and serum amylase values, compared with piratoxin-I and/or bothropstoxin-II. Histopathology of pancreas and lungs in piratoxin-I-injected rats showed interstitial oedema in both tissues, and neutrophil infiltration with acinar cell necrosis in pancreas. In conclusion, sPLA 2s induce AP in rats and the catalytic activity is not essential to induce the local effects in pancreas, although it appears to contribute partly to the remote lung injury. 相似文献
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