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1.
Six minor protein constituents (S4C10-S4C15) have been isolated from the venom of Naja melanoleuca. The complete amino acid sequence of S4C11 has been established and indicates that it is a homologue of the neurotoxins which are found in elapid venoms. The other proteins appear from the amino acid compositions to be homologues of the cyto- or cardiotoxins found in cobra venoms. Protein S4C11 has a low toxicity, failing to kill mice at an intravenous dose of 20 mug/g body weight. The sequence of the first 25 residues out of the total of 65, was determined using the automatic sequenator. The remainder of the sequence was derived with the aid of tryptic and chymotryptic peptides. The sequence showed the unusual feature of having 65 amino acid residues including 10 half-cystine residues.  相似文献   

2.
R M Kini  H J Evans 《Biochemistry》1989,28(23):9209-9215
Cardiotoxins and postsynaptic neurotoxins from snake venoms have similar primary, secondary, and tertiary structures. Cardiotoxins, however, in contrast to neurotoxins, exhibit general cytotoxicity. Comparison of the distribution of hydrophobic and charged amino acid residues in the three-dimensional structures of lytic cardiotoxins and nonlytic neurotoxins indicates the presence of a cationic site associated with a hydrophobic surface in cardiotoxins, but not in neurotoxins. A cationic site flanked by a hydrophobic site is a common structural feature shared by a wide variety of unrelated cytolysins and is predicted to determine the lytic activity of a large group of cytolysins. To determine the essential nature of the cationic site in cardiotoxin CTX-1 from Naja nigricollis crawshawii venom, we modified the positive charges of nine Lys residues to negative, neutral, or positive charges by succinylation, carbamylation, or guanidination, respectively. Circular dichroism studies indicated that these modifications did not affect the conformation of the cardiotoxin. Binding of the modified cardiotoxins to phospholipids was demonstrated by changes in the intrinsic fluorescence of native and modified CTX-1 after binding to phospholipid vesicles, and by resonance energy transfer with anthracene-phospholipid vesicles. Phospholipid binding was not affected by these modifications, but their binding preference was determined by the electrostatic interactions between the polypeptide and phospholipid. Both positively charged native and guanidinated CTX-1 showed direct lytic activity on human erythrocytes and platelets, whereas the succinylated or carbamylated derivatives did not show lytic activity. The loss of lytic activity cannot be related to conformational changes or phospholipid binding abilities of the modified cardiotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

3.
L Fryklund  D Eaker 《Biochemistry》1975,14(13):2865-2871
The complete covalent structure of a small, basic protein with cardiotoxic activity is described. This has been isolated from the venom of Naja nigricollis by gel filtration on Sephadex G-75 and gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin, molecular weight 6806 from amino acid composition, consists of 60 amino acids, cross-linked by four disulfide bridges, connecting 3-21, 14-38, 42-53, and 54-59. The protein contains one residue of tryptophan, phenylalanine, and glutamic acid, two residues of arginine and tyrosine, four residues of methionine, and nine residues of lysine. Histidine is absent. The chymotryptic peptides of the oxidized and S-carboxymethylated protein were isolated by gel filtration on Sephadex G-25 and zone electrophoresis on a cellulose column. The sequence was determined by Edman degradation, using the (manual) direct phenylthiohydantoin method and with the use of carboxypeptidase A. Disulfide pairing was determined on thermolysin cleaved peptides from the native protein. The sequence is shown to be homologous to other cardiotoxins and a lytic factor from snake venoms and also shows homology, both in sequence and disulfide pairing to neurotoxins. A partial reduction experiment in the absence of denaturing agent using 14-C-labeled iodoacetic acid as S-carboxymethylating agent shows that disulfide bonds 14-38 and 42-53 were reduced fastest followed marginally by 54-59, and then bond 3-21.  相似文献   

4.
Amino acid sequence of the nonsecretory ribonuclease of human urine   总被引:7,自引:0,他引:7  
The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
The venoms of the Naja species are known to be cytotoxic. This toxicity has been attributed to the presence of small nonenzymatic polypeptides of 60 amino acid residues, designated as cardiotoxins or cytotoxins. We investigated the cytotoxic potency of Naja nigricollis venom fractions and isolated another type of cytotoxic component which is even more potent than cardiotoxins. This cytotoxic compound, which was designated as nigexine, was purified to homogeneity and its amino acid sequence was determined. Nigexine is a basic phospholipase A2 consisting of a single chain of 118 amino acids. A detailed investigation of the cytotoxic effects on epithelial FL cells, C-13T neuroblastoma cells, and promyelocytic leukemia HL 60 cells revealed that nigexine not only altered cell viability but also prevented cell proliferation. This is a property that was specific to nigexine since other phospholipases A2 from various sources had no detectable cytotoxic activity. The cytotoxic activity of nigexine was not dependent on the presence of divalent cations, unlike its enzymatic activity. In particular, the cytotoxic activity of nigexine was identical in the presence or absence of either 2 mM Ca2+ or Sr2+, or 6 mM EDTA. We also present evidence based on chemical modifications that cytotoxic activity was not correlated with enzymatic activity. Thus, modification with parabromophenacyl bromide totally abolished the enzymatic activity of nigexine, which nevertheless retained 6-20% of the cytotoxicity of native nigexine. Conversely, treatment with cyanogen bromide gave a compound that retained 7% of the enzymatic activity of the parent molecule but was devoid of detectable cytotoxicity.  相似文献   

6.
The effects of cardiotoxin fractions from Naja naja kaouthia and Naja naja atra snake venoms and synthetic melittin peptide were examined on lipolytic activity in red blood cells and primary skeletal muscle cultures. Both native cardiotoxin fractions caused considerable production of free fatty acids in red blood cells. This production was abolished when the fractions were first treated with p-bromophenacyl bromide to reduce the venom phospholipase A2 activity contamination. In equine and human primary cultures of skeletal muscle, the N. n. kaouthia cardiotoxin (10 microM) and melittin (2 microM) caused a breakdown of phospholipids and production of free fatty acids and diacylglycerol in the absence of lysophospholipid formation. Additionally, melittin at higher concentrations (10 microM) caused triglyceride breakdown. These studies do not support the suggestion that snake venom cardiotoxins and melittin selectively activate endogenous phospholipase A2 activity. Instead, the toxins primarily activate endogenous phospholipase C activity and, in the case of melittin at high concentrations, triglyceride lipase activity.  相似文献   

7.
L Fryklund  D Eaker 《Biochemistry》1975,14(13):2860-2865
The complete amino acid sequence of a small, basic protein with cardiotoxic activity is described. This toxin, designated Naja naja F8, was isolated from the venom of Naja naja, of Cambodian origin, by gel filtration on Sephadex G-75 followed by gradient ion exchange chromatography on Bio-Rex 70. The cardiotoxin F8, molecular weight 6727 from amino acid composition, consists of 60 amino acids in a single peptide chain cross-linked by four disulfide bridges and is devoid of histidine, tryptophan, and glutamic acid. The chymotryptic and tryptic peptides from the performic acid oxidized toxin were separated by gel filtration on Sephadex G-25 and zone electrophoresis in columns of cellulose powder. The sequence was established by Edman degradation, using the direct phenylthiohydantoin method, and with the aid of carboxypetidase A, and is similar to the consequences reported for other cardiotoxins, cytotoxins, and/or lytic factors from cobra venoms, all of which show considerable homology with the functionally distinct neurotoxins.  相似文献   

8.
Incorporation of beta-sheet proteins into membrane is studied theoretically for the first time, and the results are validated by the direct experimental data. Using Monte Carlo simulations with implicit membrane, we explore spatial structure, energetics, polarity, and mode of insertion of two cardiotoxins with different membrane-destabilizing activity. Both proteins, classified as P- and S-type cardiotoxins, are found to retain the overall "three-finger" fold interacting with membrane core and lipid/water interface by the tips of the "fingers" (loops). The insertion critically depends upon the structure, hydrophobicity, and electrostatics of certain regions. The simulations reveal apparently distinct binding modes for S- and P-type cardiotoxins via the first loop or through all three loops, respectively. This rationalizes an earlier empirical classification of cardiotoxins into S- and P-type, and provides a basis for the analysis of experimental data on their membrane affinities. Accomplished with our previous simulations of membrane alpha-helices, the computational method may be used to study partitioning of proteins with diverse folds into lipid bilayers.  相似文献   

9.
P E Bougis  P Marchot  H Rochat 《Biochemistry》1986,25(22):7235-7243
The vast majority of Elapidae snake venoms, genus Naja, includes three classes of toxic polypeptides: alpha-neurotoxins, phospholipases A2, and cardiotoxins. A new experimental approach using reverse-phase high-performance liquid chromatography in particular has been developed, allowing their respective resolution, identification, and quantitation from milligram quantities of venom. First, definition of optimal chromatographic conditions for Naja mossambica mossambica toxins has been ascertained. Different column packing and solvent systems were compared for their efficiency, with particular attention to the ionic strength of the aqueous solvent. A medium-chain alkyl support (octyl) in conjunction with a volatile ammonium formate (0.15 M, pH 2.70)/acetonitrile solvent system was found to be particularly effective. All the components known until now from this venom could be resolved in a single step, and the elution order was alpha-neurotoxins, phospholipases A2, and cardiotoxins with a total recovery of absorbance and toxicity. Then, with these suitable conditions, we describe a new major cardiotoxin molecule in this venom by hydrophobic and not ionic-charge discrimination. Second, specific assays were designed to detect alpha-neurotoxin and phospholipase A2 activities in chromatographic fractions: alpha-neurotoxin activity was determined by competition for the binding of a radiolabeled alpha-neurotoxin to the acetylcholine receptor of the ray electric organ, and phospholipase A2 activity was defined by the enzymatic activity of these toxins with a fluorescent phospholipid as substrate. Finally, the applicability of these new methods to study other Naja snake venoms was demonstrated.  相似文献   

10.
Cross-linking of the unimer of glutamate dehydrogenase from beef liver (consisting of six polypeptide chains each having a molecular weight of 56000) with dimethyladipimidate and subsequent analysis by sodium dodecylsulfate electrophoresis shows predominantly the trimeric species (molecular weight 168000). Treatment with dimethylimidates of other chain length yields significantly less trimeric species indicating that the amino groups being cross-linked are within a distance of about 0.85 nm. Comparison of the molar amount of incorporated [14C]dimethyladipimidate with the number of modified amino groups (determined with trinitrobenzenesulfonic acid) shows that although 8-9 of the 34 amino groups have reacted, only 2-3 of them are involved in cross-links. Reaction with dimethylimidates inactivates the enzyme. The loss of the activity is partly concomitant to cross-linking to the trimeric species and not simply due to the modification of essential lysine residues. This is supported by the fact that, although more lysine residues react with mono-functional methylimidates, the loss of activity is reduced. Purified chymotryptic and tryptic peptides of the radioactive-labeled trimeric species were subjected to sequence analysis. Six peptides containing 75% of the total label were identified: one involves the amino-terminal residue alanine-1 and the others involve lysine-105, lysine-154, lysine-269, lysine-358 and lysine-399. Quantitative analysis of the specific radioactivity of each peptide/mol lysine leads to the conclusion that only lysine-105, lysine-154, lysine-269 and lysine-358 participate in cross-links, lysine-269 and lysine-358, respectively, being at isologous and lysine-105 cross-linked with lysine-154 at heterologous contact domains of the enzyme. A model for the planar arrangement of the trimeric species in the quaternary structure of glutamate dehydrogenase is discussed. It includes both isologous and heterologous contact areas between the polypeptide chains.  相似文献   

11.
Snake venoms are rich sources of biologically active proteins and polypeptides. Three-finger toxins are non-enzymatic proteins present in elapid (cobras, kraits, mambas and sea snakes) and colubrid venoms. These proteins contain four conserved disulfide bonds in the core to maintain the three-finger folds. Although all three-finger toxins have similar fold, their biological activities are different. A new three-finger toxin (hemachatoxin) was isolated from Hemachatus haemachatus (Ringhals cobra) venom. Its amino acid sequence was elucidated, and crystal structure was determined at 2.43 Å resolution. The overall fold is similar to other three-finger toxins. The structure and sequence analysis revealed that the fold is maintained by four highly conserved disulfide bonds. It exhibited highest similarity to particularly P-type cardiotoxins that are known to associate and perturb the membrane surface with their lipid binding sites. Also, the increased B value of hemachotoxin loop II suggests that loop II is flexible and may remain flexible until its interaction with membrane phospholipids. Based on the analysis, we predict hemachatoxin to be cardiotoxic/cytotoxic and our future experiments will be directed to characterize the activity of hemachatoxin.  相似文献   

12.
A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.  相似文献   

13.
The amino acid sequence of cytochrome c from niger-seed has been determined by sequence analysis of chymotryptic and tryptic peptides using the dansyl-phenylisothiocyanate method and by qualitative analysis of peptide composition by the dansyl method. Although the spectral ratios indicated the protein was not completely pure, no indication of impurity was found during the sequence analysis and no peptides in addition to those given here were obtained. In certain cases the alignment of peptides was by homology with other cytochromes c. Four residues in the proposed sequence, alanine-1, cysteine-25, histidine-26 and lysine-61 were identified only from peptide compositions. The amino-terminus of the protein is acetylated. The sequence contains two residues of ϵ-N-trimethyllysine.  相似文献   

14.
The cDNA encoding a novel three loop protein was cloned from cellular RNA isolated from the venom gland of Bungarus multicinctus multicinctus by RT-PCR. The mature protein has 82 amino acid residues. It shared only 25-38% similarity with some cardiotoxins and did not have sequence similarity with neurotoxins, while its cDNA was about 70% similar to both the cDNAs encoding neurotoxins and the cDNAs encoding cardiotoxins.  相似文献   

15.
A cardiotoxin-like basic polypeptide, designated as CLBP, was isolated from the venom of Naja naja atra by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The cytotoxicity toward Yoshida sarcoma cells and lethal toxicity toward mice of CLBP were both one-order lower than those of cardiotoxins and cobrotoxin, respectively. CLBP is a single polypeptide consisting of 61 amino acid residues with four intramolecular disulfide linkages. The amino acid sequence of CLBP shows a high degree of homology with those of cardiotoxins from the same venom, but differs in the 19 to 23 positions.  相似文献   

16.
Examination of the literature has revealed that regarding the amino acid sequences, cardiotoxins constitute a family of homogeneous compounds. In contrast, cardiotoxins appear heterogeneous as far as their biological and spectroscopic properties are concerned. As a result, comparison between these molecules with a view to establishing structure-activity correlations is complicated. We have therefore reviewed recent works aiming at identifying the functional site of a defined cardiotoxin, ie toxin gamma from the venom of the spitting cobra Naja nigricollis. The biological and structural properties of toxin gamma are first described. In particular, a model depicting the 3-dimensional structure of the toxin studied by NMR spectroscopy is proposed. The toxin polypeptide chain is folded into 3 adjacent loops rich in beta-sheet structure connected to a small globular core containing the 4 disulfide bonds. A number of derivatives chemically modified at a single aromatic or amino group have been prepared. The structure of each derivative was probed by emission fluorescence, circular dichroism and NMR spectroscopy. Also tested was the ability of the derivatives to kill mice, depolarize excitable cell membranes and lyse epithelial cells. Modification of some residues in the first loop, in particular Lys-12 and at the base of the second loop substantially affected biological properties, with no sign of concomitant structural modifications other than local changes. Modifications in other regions much less affected the biological properties of the toxin. A plausible functional site for toxin gamma involving loop I and the base of loop II is presented. It is stressed that the functional site of other cardiotoxins may be different.  相似文献   

17.
Allergic reactions to vespid stings are one of the major causes of IgE-mediated anaphylaxis. Vespa and Vespula venoms are closely related; Polistes venom is more distantly related and its allergens are less well studied. There is limited cross-reactivity between Polistes and the other vespid venoms because of differences in the epitopes on the allergen molecules.In this study, the major allergens of Polistes gallicus are isolated and characterized. P. gallicus venom contains four major allergens: phospholipase, antigen 5 (Ag5), hyaluronidase and protease that were characterized by mass spectrometry and specific binding to IgE. The complete amino acid sequence of Ag5 and the sequence of the N-terminal region of phospholipase were also determined. The alignment of Ag5 from P. gallicus (European species) and Polistes annularis (American species) shows an 85% identity that increases to 98% within the same subgenus. This could suggest the presence of specific epitopes on Ag5 molecule being the variations on the superficial loops. The features of the P. gallicus allergens could explain the partial cross-reactivity found between the American and European Polistes venoms, and suggest that the use of European Polistes venoms would improve the diagnostic specificity and the therapy of European patients and of North American patients sensitized by European Polistes.  相似文献   

18.
The fasciculins are a family of closely related peptides that are isolated from the venom of mambas and exert their toxic action by inhibiting acetylcholinesterase (AChE). Fasciculins belong to the structural family of three-fingered toxins from Elapidae snake venoms, which include the alpha-neurotoxins that block the nicotinic acetylcholine receptor and the cardiotoxins that interact with cell membranes. The features unique to the known primary and tertiary structures of the fasciculin molecule were analyzed. Loop I contains an arginine at position 11, which is found only in the fasciculins and could form a pivotal anchoring point to AChE. Loop II contains five cationic residues near its tip, which are partly charge-compensated by anionic side chains in loop III. By contrast, the other three-fingered toxins show full charge compensation within loop II. The interaction of fasciculin with the recognition site on acetylcholinesterase was investigated by estimating a precollision orientation followed by determination of the buried surface area of the most probable complexes formed, the electrostatic field contours, and the detailed topography of the interaction surface. This approach has led to testable models for the orientation and site of bound fasciculin.  相似文献   

19.
Loops are regions of nonrepetitive conformation connecting regular secondary structures. We identified 2,024 loops of one to eight residues in length, with acceptable main-chain bond lengths and peptide bond angles, from a database of 223 protein and protein-domain structures. Each loop is characterized by its sequence, main-chain conformation, and relative disposition of its bounding secondary structures as described by the separation between the tips of their axes and the angle between them. Loops, grouped according to their length and type of their bounding secondary structures, were superposed and clustered into 161 conformational classes, corresponding to 63% of all loops. Of these, 109 (51% of the loops) were populated by at least four nonhomologous loops or four loops sharing a low sequence identity. Another 52 classes, including 12% of the loops, were populated by at least three loops of low sequence similarity from three or fewer nonhomologous groups. Loop class suprafamilies resulting from variations in the termini of secondary structures are discussed in this article. Most previously described loop conformations were found among the classes. New classes included a 2:4 type IV hairpin, a helix-capping loop, and a loop that mediates dinucleotide-binding. The relative disposition of bounding secondary structures varies among loop classes, with some classes such as beta-hairpins being very restrictive. For each class, sequence preferences as key residues were identified; those most frequently at these conserved positions than in proteins were Gly, Asp, Pro, Phe, and Cys. Most of these residues are involved in stabilizing loop conformation, often through a positive phi conformation or secondary structure capping. Identification of helix-capping residues and beta-breakers among the highly conserved positions supported our decision to group loops according to their bounding secondary structures. Several of the identified loop classes were associated with specific functions, and all of the member loops had the same function; key residues were conserved for this purpose, as is the case for the parvalbumin-like calcium-binding loops. A significant number, but not all, of the member loops of other loop classes had the same function, as is the case for the helix-turn-helix DNA-binding loops. This article provides a systematic and coherent conformational classification of loops, covering a broad range of lengths and all four combinations of bounding secondary structure types, and supplies a useful basis for modelling of loop conformations where the bounding secondary structures are known or reliably predicted.  相似文献   

20.
Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA.  相似文献   

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