The beta-type toxin Ts II from the scorpion Tityus serrulatus: amino acid sequence determination and assessment of biological and antigenic properties.

The toxin Ts II from the venom of the Brazilian scorpion Tityus serrulatus was purified in two successive chromatographic steps. The amino acid sequence was then determined by automated Edman degradation of the reduced and S-carboxymethylated protein and of proteolytic peptides derived from it. This sequence appears to differ from that of previously characterized toxins found in this venom. However, it is identical to the recently published sequence of protein III-8 from the same venom [Possani et al., J Biol Chem 266:3178-3185, 1991], except that the C-terminus was found to be amidated. Homologies were found between the sequence of Ts II and that of other toxins from Tityus; in particular, the amino acid sequence of Ts II displays 72% sequence identity with Ts VII (also called Titx gamma). Consistent with this structural similarity, some biological properties of Ts II were found to be similar to those of Ts VII: Ts II has an intracerebroventricular LD50 of 6 ng, as compared to 0.6 ng for Ts VII; in a receptor binding assay Ts II, like Ts VII, was found to behave as a beta-type toxin and to inhibit the binding of the reference labelled toxin with a K0.5 of 5 x 10(-9) M, as compared to 7 x 10(-11) M for Ts VII. Nevertheless, Ts II is unable to bind to anti-Ts VII antibodies in radioimmunoassay experiments, indicating the non-conservation between the two toxins of at least some antigenically important residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel heat-labile phospholipid-binding protein, SVS VII, in mouse seminal vesicle as a sperm motility enhancer

SVS VII, one of seven major proteins in mouse seminal vesicle secretion, was purified to homogeneity. Neither glycoconjugate nor free thiol group was detected in the protein. The primary structure deduced from the corresponding cDNA was confirmed using amino acid sequence determination, which supported the finding that SVS VII consists of 76 amino acid residues with five disulfide bridges. Accordingly, it has a theoretical molecular mass of 8538, which was proven using the mass spectrum of SVS VII. The CD spectrum of SVS VII in 50 mm phosphate buffer at pH 7.4 appeared as one negative band arising from the beta form at 217 nm and several fine structures due to nonpeptide chromophores including a prominent band for the disulfide bond at 250 nm. This, together with the predicted secondary structures, indicated no helices but a mixture of beta form, beta turn, and unordered form in SVS VII. A cytochemical study illustrated the presence of the SVS VII-binding region on the entire surface of mouse sperm. The SVS VII-sperm binding was inhibited by the dispersed sperm lipids. The results of TLC overlay assay for the binding of (125)I-SVS VII to phospholipids and the interaction between SVS VII and phospholipid liposomes demonstrated a specific binding of this protein to both phosphatidylethanolamine and phosphatidylserine. The SVS VII-sperm binding greatly enhanced sperm motility but did not induce sperm capacitation. Heating the protein solution for 10 min at 90 degrees C unfolded the protein molecule, and the unfolded SVS VII immobilized the sperm.     

Identification of a novel family of proteins in snake venoms. Purification and structural characterization of nawaprin from Naja nigricollis snake venom

The three-dimensional structure of nawaprin has been determined by nuclear magnetic resonance spectroscopy. This 51-amino acid residue peptide was isolated from the venom of the spitting cobra, Naja nigricollis, and is the first member of a new family of snake venom proteins referred to as waprins. Nawaprin is relatively flat and disc-like in shape, characterized by a spiral backbone configuration that forms outer and inner circular segments. The two circular segments are held together by four disulfide bonds, three of which are clustered at the base of the molecule. The inner segment contains a short antiparallel beta-sheet, whereas the outer segment is devoid of secondary structures except for a small turn or 310 helix. The structure of nawaprin is very similar to elafin, a human leukocyte elastase-specific inhibitor. Although substantial parts of the nawaprin molecule are well defined, the tips of the outer and inner circular segments, which are hypothesized to be critical for binding interactions, are apparently disordered, similar to that found in elafin. The amino acid residues in these important regions in nawaprin are different from those in elafin, suggesting that nawaprin is not an elastase-specific inhibitor and therefore has a different function in the snake venom.     

Amino acid sequence of toxin VII,A β-toxin from the venom of the scorpton Tityus serrulatus

The sequence of the 61 amino acids of toxin VII, a β-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[¹⁴C] car?ymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active β-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.     

Primary structure of a protein C activator from Agkistrodon contortrix contortrix venom

The amino acid sequence of a protease, protein C activator, from Agkistrodon contortrix contortrix venom was determined. Peptide fragments obtained by chemical or enzymatic cleavage of the S-carboxymethylated protein were purified by gel filtration and reverse-phase high-performance liquid chromatography. The present study demonstrates that protein C activator from A. contortrix contortrix venom is a trypsin-type serine protease that is composed of 231 residues with a molecular weight of 25,095 for the polypeptide portion of the molecule. By analogy to the mammalian serine proteases, the catalytic triad in venom protein C activator consists of His-40, Asp-85, and Ser-177. The protein also contains three N-linked glycosylation sites at Asn-21, Asn-78, and Asn-129. The amino acid sequence of protein C activator exhibits a high degree of sequence identity with other snake venom proteases: 73% with batroxobin, 68% with flavoxobin, and 55% with Russell's viper venom factor V activator.     

Intensification of the interaction of IgG with C1q in the presence of melittin as a possible reason for anaphylaxis under the effect of bee venom

The hydrophilic head of melittin (peptide from bee venom) has the amino acid sequence which is very close to the amino acid sequence of C1q-binding site of the IgG molecule. It was shown, that melittin caused the multiple growth of the interaction of C1q with IgG monoclonal antibody. We assume, that the appearance of melittin in blood causes the spontaneous antigen-independent aggregation of IgG and C1q with the following triggering of the classical pathway of the complement cascade and origin of C3a and C5a components. This can be one of the mechanisms of anaphylaxis as an answer to bee venom.     

蛇毒的核磁共振氢谱在生物化学上的意义

每种蛇毒含有多种蛋白质组分,每种蛋白质分子有其自已的一级结构和相应的氨基酸残基的组成。因此蛇毒的氢谱是其所有组成的谱图的加和,对不同产地和种属的20多种冷冻干燥的蛇毒进行了测定,结果每种蛇毒均显示其特征的核磁共振氢谱,提示各种蛇毒的氨基酸残基组成是不同的。     

Venom exonuclease. II. Amino acid composition and carbohydrate, metal ion and lipid content in the Crotalus adamanteus venom exonuclease

Exonuclease from Crotalus adamanteus venom has only threonine as N-terminimal amono acid residue. It was examined for its amino acid composition, -SH and S-S groups. It has no free -SH groups and seven S-S bonds. The analysis of the carbohydrate residues in the enzyme proves that it is a glycoprotein. It contains neutral sugars (9.2%), amino sugars (1.9%) and ten sialic acid residues per molecule. The venom exonuclease is a metalloenzyme. This is proven by the existence of Mg2+, Zn2+ and Ca2+ and their specific role in the catalytic reactions. The enzyme contains also triacylglycerols (1.54%) and cholesterol esters (1.43%). The influence of the non-protein moieties of the exonuclease on its catalytic ability has been discussed.     

Myotoxin II from Bothrops asper (Terciopelo) venom is a lysine-49 phospholipase A2

A basic, dimeric myotoxic protein, myotoxin II, purified from Bothrops asper venom has a similar molecular weight and is immunologically cross-reactive with antibodies raised to previously isolated B. asper phospholipases A2, except that it shows only 0.1% of the phospholipase activity against L-alpha-phosphatidylcholine in the presence of Triton X-100. Its 121 amino acid sequence, determined by automated Edman degradation, clearly identifies it as a Lys-49 phospholipase A2. Key amino acid differences between myotoxin II and phospholipase active proteins in the Ca2(+)-binding loop region, include Lys for Asp-49, Asn for Tyr-28, and Leu for Gly-32. The latter substitution has not previously been seen in Lys-49 proteins. Other substitutions near the amino terminus (Leu for Phe-5 and Gln for several different amino acids at position 11) may prove useful for identifying other Lys-49 proteins in viperid and crotalid venoms. Myotoxin II shows greater sequence identity with other Lys-49 proteins from different snake venoms (Agkistrodon piscivorus piscivorus, Bothrops atrox, and Trimeresurus flavoviridis) than with another phospholipase A2 active Asp-49 molecule isolated from the same B. asper venom. This work demonstrates that phospholipase activity per se, is not required in phospholipase molecules for either myotoxicity or edema inducing activities.     

Conformational flexibility of a scorpion toxin active on mammals and insects: a circular dichroism study

Three scorpion toxins have been analyzed by circular dichroism in water and in 2,2,2-trifluoroethanol (TFE) solutions. These toxins were chosen because they are representative of three kinds of pharmacological activities: (1) toxin AaH IT2, an antiinsect toxin purified from the venom of Androctonus australis Hector, which is able to bind to insect nervous system preparation, (2) toxin Css II, from the venom of Centruroides suffusus suffusus, which is a beta-type antimammal toxin capable of binding to mammal nervous system preparation, and (3) the toxin Ts VII from the venom of Tityus serrulatus, which is able to bind to both types of nervous systems. In order to minimize bias, CD data were analyzed by a predictive algorithm to assess secondary structure content. Among the three molecules, Ts VII presented the most unordered secondary structure in water, but it gained in ordered forms when solubilized in TFE. These results indicated that the Ts VII backbone is the most flexible, which might result in a more pronounced tendency for this toxin molecule to undergo conformational changes. This is consistent with the fact that it competes with both antiinsect and beta-type antimammal toxins for the binding to the sodium channel.     

Bovine factor VII. Its purification and complete amino acid sequence

A modified method for purification of blood clotting factor VII from bovine plasma was developed, and its complete amino acid sequence was established. The isolated factor VII was activated with factor XIIa, and the resulting two-chain factor VII (factor VIIa) was reduced and S-pyridylethylated or S-aminoethylated. The amino acid sequences of the S-alkylated heavy and light chains were determined by sequencing the fragments obtained from enzymatic and chemical cleavages. Fast atom bombardment mass spectrometry was also used to establish the COOH-terminal sequence of the heavy chain. The light chain consists of 152 residues with one carbohydrate chain at Asn145, and 11 gamma-carboxyglutamic acid residues are found within the NH2-terminal 35 residues. The light chain contains 0.2-0.3 mol of beta-hydroxyaspartic acid/mol of protein, indicating that an aspartic acid residue in bovine factor VII is incompletely hydroxylated. Moreover, a pentapeptide, Ala-Ser*-Ser-Pro-Cys (positions 51-55), isolated from an enzymatic digest of the light chain, contained an unknown serine derivative, but its structure is still unclear. On the other hand, the heavy chain is composed of 255 residues and one asparagine-linked carbohydrate chain at Asn203. Bovine factor VII, with a total of 407 residues, has 71% sequence identity with the human molecule (406 residues) predicted from the cDNA sequence (Hagen, F. S., Gray, C. L., O'Hara, P., Grant, F. J., Saari, G. C., Woodbury, R. G., Hart, C. E., Insley, M., Kisiel, W., Kurachi, K., and Davie, E. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2412-2416).     

Localization of the amino acid substitution site in a fast migrating variant of human serum albumin

Albumin Mi/Fg is an Italian genetic variant of human serum albumin arising from a Lys----Glu substitution which has been located in a CNBr fragment (CNBr VII) corresponding to the -COOH terminal portion of the molecule [(1984) J. Chromatogr. 298, 336-344]. Tryptic peptides of CNBr VII from normal and Mi/Fg albumin have been purified by reverse-phase high-performance liquid chromatography (RP-HPLC) and submitted to comparative structural studies. The amino acid sequence of the tryptic peptide of Mi/Fg variant that differs from the corresponding fragment of the normal serum albumin shows that the Lys----Glu substitution responsible for this variant is located at position 573. This region of the albumin molecule is involved in the binding of long chain fatty acids.     

A phospholipase A2 with anticoagulant activity. I. Isolation from Vipera berus venom and properties.

An anticoagulant protein has been isolated by DEAE cellulose chromatography and gel filtration from the venom of the Vipera berus orientale (Eastern Europe). Purification has been completed by elution on carboxymethyl cellulose with continuous gradient at constant pH. The inhibitor of coagulation was separated from the other venom enzymes, e.g. procoagulant, fibrinogenolytic, aminoesterase and amino acid oxidase activities. It was also separated from other phospholipase components which were not related to the anticoagulant property. The inhibitor appeared as a simgle polypeptidic chain protein, formed by 119 amino acid residues, with a molecular weight of 13400 and an isoelectric point of 9.2. At low saline molarity, a monomer-trimer transition of this protein was observed. Both forms had the same amino acid composition. There were six disulfide bridges without free SH groups per phospholipase molecule. Deprived of any proteolytic activity, the clotting inhibitor displayed a high phospholipase activity in the presence of calcium. Activity did no appear with EDTA buffer deprived of cation. Finely dispersed micellar suspensions were found suitable for obtaining the highest phospholipase activity. High sodium cholate concentration or methanol/chloroform/ether solvent were effective without loss of enzymatic activity. As characteristis of phospholipase A2 (EC 3.1.1.4), the degradation products identified on thin-layer chromatography induced hemolysis of human erythrocytes. The apparent Km value 1.25 - 10(-3) M was determined on phosphatidylcholine isolated from ovolecithin. This purified berus inhibitor would be of value for investigating the involvement of phospholipids in the clotting mechanism.     

Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom.

The complete amino acid sequence of fibrolase, a fibrinolytic enzyme from southern copperhead (Agkistrodon contortrix contortrix) venom, has been determined. This is the first report of the sequence of a direct-acting, nonhemorrhagic fibrinolytic enzyme found in snake venom. The majority of the sequence was established by automated Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. The amino-terminus is blocked by a cyclized glutamine (pyroglutamic acid) residue, and the sequence of this region of the molecule was determined by mass spectrometry. Fibrolase is composed of 203 residues in a single polypeptide chain with a molecular weight of 22,891, as determined by the sequence. Its sequence is homologous to the sequence of the hemorrhagic toxin Ht-d of Crotalus atrox venom and with the sequences of two metalloproteinases from Trimeresurus flavoviridis venom. Microheterogeneity in the sequence was found at both the amino-terminus and at residues 189 and 192. All six cysteine residues in fibrolase are involved in disulfide bonds. A disulfide bond between cysteine-118 and cysteine-198 has been established and bonds between cysteines-158/165 and between cysteines-160/192 are inferred from the homology to Ht-d. Secondary structure prediction reveals a very low percentage of alpha-helix (4%), but much greater beta-structure (39.5%). Analysis of the sequence reveals the absence of asparagine-linked glycosylation sites defined by the consensus sequence: asparagine-X-serine/threonine.     

Primary structure of the antihemorrhagic factor in serum of the Japanese Habu: a snake venom metalloproteinase inhibitor with a double-headed cystatin domain.

The complete amino acid sequence of an antihemorrhagic factor, HSF, in the serum of the Japanese Habu snake, Trimeresurus flavoviridis, has been determined. The protein is composed of 323 amino acid residues and contains three asparagine-linked oligosaccharide chains at positions 123, 185, and 263. The molecule contains two copies of the cystatin domain in the N-terminal portion up to position 240, and these domains show a remarkable sequence homology (about 50%) to those of plasma glycoproteins such as alpha 2-HS (human) and fetuin (bovine) and to a lesser extent to that of HRG (human). The amino acid sequence of the noncystatin region towards the C-terminus is unique, showing no significant homology with those of the corresponding regions of alpha 2-HS and fetuin. In spite of the presence of cystatin domains, HSF does not inhibit cysteine proteinases such as papain and cathepsin B but does inhibit several metalloproteases in Habu venom. The results suggest that HSF is the first protein found to be functionally related to metalloproteinase inhibitors among the structurally homologous proteins with a double-headed cystatin domain, and is a member of a novel family (family 4) with divergent functions of the cystatin superfamily proteinase inhibitors. Although HSF possesses similar physicochemical properties to those of oprin, a snake venom metalloproteinase inhibitor with antihemorrhagic activity isolated from opossum serum [Catanese & Kress (1992) Biochemistry 31, 410-418], its primary structure is strikingly different from that of oprin.     

A bradykinin-potentiating peptide (peptide K12) isolated from the venom of Egyptian scorpion Buthus occitanus

A nontoxic peptide with bradykinin-potentiating activity was isolated from the dialyzed venom of the scorpion Buthus occitanus by reverse-phase high performance liquid chromatography (RP-HPLC). The pharmacological activity of the peptide was bioassayed by its ability to potentiate added bradykinin (BK) on the isolated guinea pig ileum as well as the isolated rat uterus for contraction. Moreover, the peptide potentiates in vivo the depressor effect of BK on arterial blood pressure in the normotensive anesthetized rat. Chemical characterization of the peptide was also performed. The amino acid composition of the peptide showed 21 amino acid residues per molecule including three proline residues. The amino acid sequence of the purified peptide was confirmed by mass spectrometry. Either N- or C-terminal ends were free. The sequence does not show a homology with bradykinin-potentiating peptides isolated from either scorpion or snake venoms. Furthermore, we did not find a significant sequence homology between the sequence of the isolated peptide and any of proteins or peptides in GenPro or NBRF data banks. The peptide also inhibited angiotensin-converting enzyme (ACE), and could not serve as substrate for the enzyme. It could be concluded that the mechanism of bradykinin-potentiating peptide (BPP) activity may be due to ACE inhibition.     

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.     

Differential expression and geographic variation of the venom phospholipases A2 of Calloselasma rhodostoma and Trimeresurus mucrosquamatus

To investigate the geographic variations in venoms of two medically important pitvipers, we have purified and characterized the phospholipases A2 (PLA2s) from the pooled venoms of Calloselasma rhodostoma from Malaysia, Thailand, Indonesia, and Vietnam, as well as the individual venom of Trimeresurus mucrosquamatus collected from both North and South Taiwan. Enzymatic and pharmacological activities of the purified PLA2s were also investigated. The complete amino acid sequences of the purified PLA2s were determined by sequencing the corresponding cDNAs from the venom gland and shown to be consistent with their molecular weight data and the N-terminal sequences. All the geographic venom samples of C. rhodostoma contain a major noncatalytic basic PLA2-homolog and two or three acidic PLA2s in different proportions. These acidic PLA2s contain Glu6-substitutions and show distinct inhibiting specificities toward the platelets from human and rabbit. We also found that the T. mucrosquamatus venoms from North Taiwan but not those from South Taiwan contain an Arg6-PLA2 designated as TmPL-III. Its amino acid sequence is reported for the first time. This enzyme is structurally almost identical to the low- or nonexpressed Arg6-PLA2 from C. rhodostoma venom gland, and thus appears to be a regressing venom component in both of the Asian pitvipers.     

L-氨基酸氧化酶的研究进展

L-氨基酸氧化酶(L-amino acid oxidase, LAAO)能特异性催化L-氨基酸氧化脱氨,生成α-酮酸、氨和H₂O₂。该酶分布较广,其中蛇毒源LAAO是该类酶中研究最为深入的一类,近年来,越来越多的非蛇毒源LAAO被发现和报道,现对蛇毒源和非蛇毒源LAAO的研究进展进行了综述。现有研究表明,不同物种来源的LAAO,其底物选择性、等电点、稳定性等理化性质不尽相同;虽对其结构的研究还较少,但现有的研究表明蛇毒源和非蛇毒源LAAO的结构都含有FAD结合结构域、底物结构域和螺旋结构域;研究已发现不同来源的LAAO体外具有多种不同的生物学功能,而这些生物学功能多数是由于其产物H₂O₂作用的结果;对LAAO异源表达的研究较少且都不甚成功,可能是由于其需要进行翻译后修饰。