蝶蛹金小蜂毒液蛋白不同提取分离方法的比较

为了建立蝶蛹金小蜂Pteromalus puparum毒液抑制寄主血细胞免疫活性组分合适的分离纯化方法,就等电点沉淀法、乙醇沉淀法、75%硫酸铵沉淀法、75%硫酸铵沉淀法+40℃加热处理法,以及75%硫酸铵沉淀法分别与3种不同滤膜的分子大小截留法的组合等7种方法对毒液蛋白分离效果及活性的影响进行了比较。结果表明：等电点沉淀法获得的组分抑制寄主菜粉蝶Pieris rapae离体血细胞延展和包囊的活性最强,乙醇沉淀法次之,75%硫酸铵沉淀法最弱。从蛋白组分的SDS-PAGE图谱来看,等电点沉淀法获得毒液组分相对最纯,仅有3条主要谱带,分子量大小在45～116.2 kDa范围内;乙醇沉淀法次之,有5条主要谱带,分子量大小在24～116.2 kDa范围内;硫酸铵沉淀法的谱带组成与毒液蛋白粗提液相似。3种分子大小截留法获得的毒液组分的活性分析表明,强活性组分分子量大小可能都大于100 kDa。综合认为,7种方法中以等电点沉淀法提取分离蝶蛹金小蜂毒液蛋白相对为最适。     

Molecular components and toxicity of the venom of the solitary wasp, Anoplius samariensis

The solitary spider wasp, Anoplius samariensis, is known to exhibit a unique long-term, non-lethal paralysis in spiders that it uses as a food source for its larvae. However, neither detailed venom components nor paralytic compounds have ever been characterized. In this study, we examined the components in the low molecular weight fraction of the venom and the paralytic activity of the high molecular weight fraction. The major low molecular weight components of the venom were identified as gamma-aminobutyric acid and glutamic acid by micro-liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectrometry analysis. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass analysis revealed that the A. samariensis venom contained the various proteins with weights of 4-100 kDa. A biological assay using Joro spiders (Nephila clavata) clearly showed that the high molecular weight fraction of the venom prepared by ultrafiltration exerted as potent non-lethal long-term paralysis as the whole venom, whereas the low molecular weight fraction was devoid of any paralytic activity. These results indicated that several venomous proteins in the high molecular weight fraction are responsible for the paralytic activity. Furthermore, we determined the primary structure of one component designated As-fr-19, which was a novel multiple-cysteine peptide with high sequence similarity to several sea anemone and snake toxins including dendrotoxins, rather than any insect toxic peptides identified so far. Taken together, our data showed the unprecedented molecular and toxicological profiles of wasp venoms.     

马氏蝎毒毒素的分离纯化及其对红细胞膜作用的初步研究

本文用山东产马氏蝎(Buthus martensii kashi)粗毒为材料,经SephadexG-50和Sp-Sephadex C-25二次柱层析,分离纯化获得三个毒峰部分,毒性比粗毒分别提高40—100倍。 纯度鉴定表明三个毒峰的聚丙烯酰胺凝胶电泳和等电聚焦电泳均为一条带,等电点分别为8.7,9.1,9.1,分子量用SDS-不连续聚丙烯酰胺凝胶电泳测定分别为6,600,5,000和8,500。对纯化蝎毒毒素的氨基酸组分也作了分析。 蝎毒毒素对人红细胞膜作用的初步探索结果表明:它使人红细胞膜的Na.K-ATP酶活性和膜脂流动性有所降低。     

Bothrops moojeni venom kills Leishmania spp. with hydrogen peroxide generated by its L-amino acid oxidase

Leishmaniasis is an endemic tropical disease in South America, with few therapeutic approaches. Snake venoms are complex protein mixtures with biological actions that could be used as tools for drug development. Here we show that Bothrops moojeni crude venom presented a killing effect in vitro against Leishmania spp. promastigotes, but not with amastigotes, as determined by a viability assay using the mitochondrial oxidative function. Purification of active fractions from crude venom was performed by molecular exclusion and ion exchange chromatography. Anti-Leishmania and l-amino acid oxidase (L-AAO, EC.1.4.3.2.) activities co-eluted in the same fractions. The molecular weight of the active enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa by SDS--PAGE, with a 4.8 isoelectric point. Using substrate subtraction and catalase for scavenging, the action of L-AAO was demonstrated to be hydrogen-peroxide-dependent.     

Intramitochondrial location and some characteristics of chicken liver aspartate aminotransferase

Chicken liver mitochondrial aspartate aminotransferase was found to be located in the intermembrane space and bound to the inner mitochondrial membrane. Purification of two mitochondrial fractions containing aspartate aminotransferase activity was performed. Both fractions showed similar chromatographic behaviour and identical isoelectric point and molecular weight values. There were no significant differences in the general kinetic mechanism, Km values, substrates inhibition and effect of various anions on the activity of mitochondrial aspartate aminotransferase purified from both fractions.     

Characterization and biochemical analyses of venom from the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae)

During parasitism, the ectoparasitic wasp Nasonia vitripennis (Walker) (Hymenoptera: Pteromalidae) induces a developmental arrest in host pupae that is sustained until the fly is either consumed by developing larvae or the onset of death. Bioassays using fluids collected from the female reproductive system (calyx, alkaline gland, acid gland, and venom reservoir) indicated that the venom gland and venom reservoir are the sources of the arrestant and inducer(s) of death. Infrared spectroscopic analyses revealed that crude venom is acidic and composed of amines, peptides, and proteins, which apparently are not glycosylated. Reversed phase high performance liquid chromatography (HPLC) and sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the proteinaceous nature of venom and that it is composed mostly of mid to high molecular weight proteins in the range of 13 to 200.5 kilodaltons (kDa). Ammonium sulfate precipitation and centrifugal size exclusion membranes were used to isolate venom proteins. SDS-PAGE protein profiles of the isolated venom fractions displaying biological activity suggest that multiple proteins contribute to arresting host development and eliciting death. Additionally, HPLC fractionation coupled with use of several internal standards implied that two of the low molecular weight proteins were apamin and histamine. However, in vitro assays using BTI-TN-5B1-4 cells contradict the presence of these agents.     

Purification of glutamate dehydrogenase from the rabbit liver and study of its major physico-chemical properties

Highly purified preparations of glutamate dehydrogenase were obtained from mitochondrial and cytoplasmic fractions of rabbit liver by affinity chromatography on CL-Sepharose 4B modified by adenosine diphosphate. Some physico-chemical properties of the purified enzymes (e. g., specific activity, molecular weight, quaternary structure, stability against denaturating effect of urea, pH optimum of catalyzed reactions, Km values for substrates and coenzymes) were found to be identical. The sole difference was detected in the ability of enzyme preparations to be activated by adenosine diphosphate. The activation of the cytoplasmic enzyme is 160%, that of mitochondrial glutamate dehydrogenase is 230-240% under the same conditions.     

Proteomic analysis of Latrodectus tredecimguttatus venom for uncovering potential latrodectism-related proteins

Black widow spider is one of the most poisonous spiders in the world. Up to now, there have been few systematic analyses of the spider venom components, and the mechanism of action of the venom has not been completely understood. In this work, we employed combinative proteomic strategy to analyze the venom collected from living adult spider Latrodectus tredecimguttatus by electrical stimulation. The experiments demonstrated that the venom is primarily composed of high molecular weight proteins and has high abundance proteins around 100 kDa. The content of peptides and proteins with low molecular weight is low. A total of 75 nonredundant venom proteins with distinct function were unambiguously identified. Besides the known black widow spider venom proteins including latrotoxins, a variety of hydrolases and other proteins with special activity were found in the venom, such as proteinase, phospholipase, phosphatase, nuclease, fucolectin, venom allergen antigen 5-like protein and trypsin inhibitor, and so on. Their possible biological actions and relationship with latrodectism were discussed. The results help to understand the complexity and action mechanism of L. tredecimguttatus venom.     

Parasitoid wasp sting: a cocktail of GABA, taurine, and beta-alanine opens chloride channels for central synaptic block and transient paralysis of a cockroach host

The wasp Ampulex compressa injects venom directly into the prothoracic ganglion of its cockroach host to induce a transient paralysis of the front legs. To identify the biochemical basis for this paralysis, we separated venom components according to molecular size and tested fractions for inhibition of synaptic transmission at the cockroach cercal-giant synapse. Only fractions in the low molecular weight range (<2 kDa) caused synaptic block. Dabsylation of venom components and analysis by HPLC and MALDI-TOF-MS revealed high levels of GABA (25 mM), and its receptor agonists beta-alanine (18 mM), and taurine (9 mM) in the active fractions. Each component produces transient block of synaptic transmission at the cercal-giant synapse and block of efferent motor output from the prothoracic ganglion, which mimics effects produced by injection of whole venom. Whole venom evokes picrotoxin-sensitive chloride currents in cockroach central neurons, consistent with a GABAergic action. Together these data demonstrate that Ampulex utilizes GABAergic chloride channel activation as a strategy for central synaptic block to induce transient and focal leg paralysis in its host.     

Purification of a nontoxic phospholipase A2 from the venom of Indian krait (Bungarus caeruleus).

A nontoxic phospholipase A2 was purified from the venom of Indian krait (Bungarus caeruleus) by a four-step procedure involving electrophoresis, gel filtration and ion-exchange chromatography. The recovery of the enzyme activity was 37% and the purified preparation was 38 times as active as the crude venom. The purified enzyme had a molecular weight of 12,500 and the optimum pH of 7.2. The enzyme showed higher specificity toward phosphatidylethanolamine than phosphatidylcholine. The preparation was not very labile to heat and its activity was dependent on the presence of divalent cations, calcium ions being the most effective activators. The enzyme was completely inhibited by iodoacetic acid but showed high stability against 8 M urea. Purified phospholipase A2 was nontoxic at an iv dose of 5 microgram/g mouse. The high specific activity, the high yield and the nontoxic nature of the enzyme indicate that the major form of phospholipase A2 in Bungarus caeruleus venom is not associated with any toxicity and has properties somewhat similar to that of phospholipase A2 from some other venoms.     

Tarantula (Eurypelma californicum) venom, a multicomponent system

The venom of the tarantula Eurypelma californicum was analysed biochemically, the components were isolated and characterized. The pH value of the crude venom is 5.3 +/- 0.3. After dilution with distilled water, UV-absorption spectra showed a single maximum at 258 nm (pH ca. 7.0). A second maximum at 328 nm emerged above pH 8.0. Protein concentration of the venom is ca. 65 mg/ml. After Coomassie staining SDS-PAGE patterns show three major bands with apparent molecular masses around 40 kDa, 4.3 kDa and 1.3 kDa besides some weak high molecular protein bands. The following low-molecular mass constituents were determined in the crude venom: ATP, ADP, AMP, glutamic acid, aspartic acid, gamma-aminobutyric acid, glucose and the ions potassium, sodium, calcium, magnesium and chloride; the osmolality was 361 micro0smol/ml. The LD50 value for female cockroaches was 0.15 microliters venom per g body weight and for male cockroaches 0.4 microliters venom per g body weight. Separation of the crude venom by gel chromatography yielded four elution peaks. Peak I contains the enzyme hyaluronidase. The activity is 200-900 U/microliters. Peak II contains a mixture of toxic peptides. Peak III contains the 1.3-kDa components of SDS-PAGE and peak IV mainly contains ATP. Venom proteins including the enzyme hyaluronidase were precipitated by 5% trichloroacetic acid. The supernatant was separated by HPLC into 13 fractions. Fraction 1 contains glutamic acid, aspartic acid, gamma-aminobutyric acid and ATP; fraction 2 contains ATP, ADP and AMP as well as a component 2' visible in SDS-PAGE as 1.3-kDa band and consisting of spermine and tryptophan; fraction 3 contains ATP and an unknown component 3'; fractions 4-6 also show a 1.3-kDa band in SDS-PAGE, fraction 4 being tyrosylspermine and fractions 5 and 6 containing compounds of spermine and aromatic molecules; fraction 7 contains a peptide which lacks aromatic amino acids, it was sequenced from the N-terminus; fractions 8-13 contain very similar toxic peptides. The peptides in fractions 11 and 12, labeled ESTX for Eurypelma spider toxin, were cleaved with different enzymes and sequenced. They differ in one amino acid in position 26. Homologies with scorpion toxins and with a toxin of the spider Segestria florentina were found.     

Separation and purification of Echis coloratus venom and some biological and biochemical effects of the proteins

Crude venom of Echis coloratus was separated into seven protein fractions using 7% preparative native polyacrylamide gel electrophoresis. The effect of crude venom and seven venom protein fractions (F1-F7) from Echis coloratus on key metabolic activities of fibroblast cultures was investigated. Confluent cultures were incubated with the venom proteins for 3 h at 37 degrees C. The specific activity of phosphofructokinase, was significantly lowered upon incubation with the crude venom and with fractions 2, 3, 4 and 6. Citrate synthase activity was significantly lowered by the crude venom and by fractions 2 and 3. Glycogen phosphorylase activity was significantly increased by the crude venom and by fractions 2, 3, 4 and 6 leading to a significant concurrent drop in glycogen content. Creatine kinase activity was significantly increased by the crude venom and by fractions 3, 4, 5 and 6. Cellular ATP levels rose significantly upon incubation with the crude venom and with fractions 3, 4, 5 and 6. Incubation of cell sonicates with all the venom proteins did not significantly alter the activity or content of any of the studied parameters.     

Presence of a low molecular weight inhibitor of succinate-supported cholesterol side chain cleavage in rat ovaries

1. Low molecular weight fractions (mol. wt. 3500-10 000) prepared from cytosols of luteinized rat ovaries inhibited succinate-supported cholesterol side chain cleavage by intact ovarian mitochondria utilizing endogenous or exogenous sterol as substrate. 2. The low molecular weight fractions inhibited steroid secretion by collagenase-dispersed ovarian cells stimulated with lutropin or dibutyryl cyclic AMP. 3. Steroidogenesis by intact mitochondria incubated with NADPH was enhanced by the low molecular weight ovarian fraction, but cholesterol side chain cleavage carried out by sonicated mitochondria incubated with NADPH was unaffected. 4. Succinate-supported mitochondrial respiration was stimulated by the low molecular weight factor, apparently by uncoupling of oxidative phosphorylation. The uncoupling seems to be the mechanism by which steroid synthesis is inhibited. 5. The low molecular weight factor was heat-labile and not extracted by activated charcoal. Similar heat-labile material capable of inhibiting succinate-supported mitochondrial steroid synthesis was not found in low molecular weight fractions prepared from rat kidney, liver, spleen, brain, plasma and bovine corpus luteum. 6. Treatment of rats with cycloheximide 1 h before killing resulted in a reduction of inhibitory activity in ovarian low molecular weight cytosolic fractions. 7. We conclude that ovarian cytosols contain a low molecular weight factor, presumably a protein, which inhibits mitochondrial cholesterol side chain cleavage by uncoupling oxidative phosphorylation. The physiological function of this factor remains to be determined.     

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.     

Proteolytic, procoagulant activity and electrophoretic characterization of the proteins of 2 toxic extracts of Loxosceles laeta venom

Some biochemical properties and proteic components of the brown spider (Loxos celes laeta) venom were studied. The electrophoretic profiles of glandular venom and venom obtained through electrical stimulation were compared using two electrophoretic systems. The first, using a polyacrylamide gel with SDS in tubes, and the second, using an acrylamide gradient on slides. The glandular venom presented 20 and 35 bands respectively, while the venom obtained through electrical stimulation presented 19 and 24 bands. The molecular weight of the proteins detected ranged from 13.5 Kd to 220 Kd. A thermolabil proteolitic activity of casein was detected, and was optimum at pH 9. The effects of the divalent ions, calcium and magnesium, as well as that of chelating agents upon the proteolytic activity of the venom were analyzed. The venom had a procoagulant effect upon citrated human plasma, and was not able to activate the Factor X of the coagulation system in vitro.     

Hemorrhagic, coagulant and fibrino(geno)lytic activities of crude venom and fractions from mapanare (Bothrops colombiensis) snakes

Bothrops colombiensis venom from two similar geographical locations were tested for their hemostatic functions and characterized by gel-filtration chromatography and SDS-PAGE electrophoresis. The snakes were from Caucagua and El Guapo towns of the Venezuelan state of Miranda. Fibrino(geno)lytic, procoagulant, hemorrhagic, lethal activities, gel-filtration chromatography and SDS-PAGE profiles were analyzed and compared for both venoms. The highest hemorrhagic activity of 5.3 mug was seen in El Guapo venom while Caucagua venom had the lowest LD(50) of 5.8 mg/kg. Both venoms presented similar thrombin-like activity. El Guapo showed a factor Xa-like activity two times higher than Caucagua. Differences were observed in kallikrein-like and t-PA activities, being highest in El Guapo. Caucagua venom showed the maximum fibrin lysis. Both crude venom runs on Sephadex G-100 chromatography gave fraction SII with the high fibrinolytic activity. Proteases presented in SII fractions and eluted from Benzamidine-Sepharose (not bound to the column) provoked a fast degradation of fibrinogen alpha chains and a slower degradation of beta chains, which could possibly be due to a higher content of alpha fibrinogenases in these venoms. The fibrinogenolytic activity was decreased by metalloprotease inhibitors. The results suggested that metalloproteases in SII fractions were responsible for the fibrinolytic activity. The analysis of samples for fibrin-zymography of SII fractions showed an active band with a molecular mass of approximately 30 kDa. These results reiterate the importance of using pools of venoms for antivenom immunization, to facilitate the neutralization of the maximum potential number of toxins.     

Properties of nerve growth factor from the venom of Bothrops atrox.

A glycoprotein fraction islated in poor yield (approx. 0.04%) from the venom of Bothrops atrox contained nerve growth factor. The material had biological activity, stability, the property of anomalous adsorption onto surfaces, apparent molecular weight and sub-unit structure that were similar to those for nerve growth factor that had been previously purified from the venom of Vipera russelli. Antiserum raised against nerve growth factor from Vipera russelli showed definite cross-reactivity with either the material from Bothrops atrox or a nerve growth factor-containing fraction from the venom of Ancistrodon piscivorus piscivorus, while antiserum against nerve growth factor from mouse had little effect on any of these preparations.     

Localization, isolation and characterization of Mn-superoxide dismutase in bovine adrenocortical cells

Cyanide-insensitive superoxide dismutase activity was present in both cytosol (26%) and mitochondrial (64%) fractions of bovine adrenal cells. The cyanide-insensitive superoxide dismutase was isolated from the mitochondrial fraction. It contained 2.2 g atoms of manganese per mol of enzyme. The enzyme had a molecular weight of 82,000 and a subunit molecular weight of 22,000. The isoelectric point, amino acid composition, and spectra of visible and ultraviolet regions were similar to those of the Mn-superoxide dismutase from the other mitochondria.