Crystallization and preliminary crystallographic study of stonustoxin, a protein lethal factor isolated from the stonefish (Synanceja horrida) venom

Crystals of stonustoxin have been obtained and diffract to 3.4 A resolution. Stonustoxin is a protein lethal factor isolated from the venom of the stonefish, Synanceja horrida. The crystals belong to the tetragonal space group P422, with unit cell constants a = b = 109.0 A, c = 245.7 A. A native stonustoxin molecule has two subunits, designated alpha and beta, respectively, and there is one stonustoxin molecule per asymmetric unit.     

Beta-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms

Beta-conglycinin consisting of six major isomers (designated B1- to B6-conglycinin) was dissociated and fractionated on columns of DEAE- and CM-Sephadex in buffers containing 6 M urea. Three major (alpha, alpha' and beta) and one minor (gamma) subunits were isolated and further characterized by gel electrophoresis and gel electrofocusing. Gel electrophoresis in urea and in sodium dodecyl sulfate, and gel filtration in 6 M guanidine hydrochloride gave a molecular weight of 57 000 for alpha, alpha' subunits; and 42 000 for beta and gamma subunits. The isoelectric points of the isolated subunits, measured by disc gel electrofocusing, were as follows: alpha, 4.90; alpha', 5.18; beta, 5.66-6.00. On gel electrofocusing, beta subunit showed four microheterogeneous components; three of them comprised 95% of the total beta subunit. Leucine and valine were the N-terminal amino acids of beta and alpha alpha' subunits, respectively. The isolated subunits contained mannose and glucosamine in varying quantities. Two carbohydrate moieties were calculated for one mole of alpha, alpha' subunits; and one carbohydrate moiety for the beta subunit. Considerable similarity in the amino acid composition of alpha and alpha' subunits was observed. The beta subunit was devoid of cysteine and methionine; and in comparison with alpha, alpha' subunits, had a higher content of hydrophobic amino acids. The isolated subunits exhibited antigen-antibody reaction with antisera to the native beta-conglycinin. Each of them was partglycinins. The alpha and alpha' subunits were in addition identical with each other and with B5-, B6-conglycinins. They were immunologically unrelated with beta subunit. The recovery of immuno-properties from the individual subunits may be attributed to the reconstruction of the three-dimensional structure upon removal of denaturing reagents.     

Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center.     

Amino-acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus)

The amino acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan, has been determined. The beta 1 isosubunit, which is neither toxic nor enzymatically active on its own, consists of a single polypeptide chain of 118 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin beta 1 isosubunit is closely homologous to the taipoxin alpha and gamma subunits and to enzymatically active pancreatic and elapid snake venom phospholipases A2.     

Isolation and characterization of the receptor on human neutrophils that mediates cellular adherence

The receptor on human neutrophils (polymorphonuclear leukocytes or PMN) that mediates cellular adherence has been purified from the peripheral blood PMN obtained from an individual with chronic myelogenous leukemia (CML). This receptor consists of two noncovalently associated subunits, designated alpha M (Mac-1 alpha, CD11b) (Mr = 170,000) and beta (Mac-1 beta, CDw18) (Mr = 100,000), respectively, which are identical on normal and CML PMN. The subunits were purified by monoclonal antibody 60.1-Sepharose (anti-alpha M) affinity chromatography and separated in 5-nmol quantities by high pressure liquid chromatography on a TSK-4000 gel filtration column. Subunits were characterized by amino acid composition, NH2-terminal amino acid sequence, and carbohydrate content. The NH2-terminal sequence of the human PMN alpha M subunit contains regions of homology with the human platelet glycoprotein IIb alpha. We conclude that nanomole amounts of individual alpha M and beta subunits of the receptor on human PMN that mediates cellular adherence can be isolated and separated using CML PMN.     

Lebecetin, a potent antiplatelet C-type lectin from Macrovipera lebetina venom

A novel C-type lectin protein (CLP), lebecetin, was purified to homogeneity from the venom of Macrovipera lebetina by gel filtration on a Sephadex G75 column and ion exchange chromatography on Mono S column. Lebecetin is a basic protein with a pHi=9.9 and migrates in SDS-PAGE as a single band or two distinct bands under nonreducing and reducing conditions, respectively. These results are further confirmed by MALDI-TOF mass spectrometry that indicates a molecular mass of 29779 Da for native lebecetin and molecular masses of 15015 and 16296 Da for alpha and beta subunits, respectively. The N-terminal amino acid sequences of lebecetin subunits show a high degree of similarity with those of C-type lectin-like proteins. In addition, functional studies showed that lebecetin has a potent inhibitory effect on platelet aggregation induced by thrombin in a concentration-dependent manner. In contrast, no inhibitory effect is observed when platelets are exposed to thromboxane A2 (TxA2) mimetic (U46619) or arachidonic acid. Moreover, there was no effect either on blood coagulation or A, B and O washed human erythrocytes agglutination. Furthermore, flow cytometric analysis revealed that fluoro-isothiocyanate (FITC)-labelled lebecetin bound to human formalin fixed platelets in a saturable and concentration manner and this binding was specifically prevented by anti-glycoprotein Ib (GPIb) mAb. These observations suggest that lebecetin is a C-type lectin-like protein that selectively binds to platelet GPIb.     

Subunit structure of a potent platelet-activating glycoprotein isolated from the venom of Crotalus durissus cascavella

The potent platelet-activating factor isolated from the venom of Crotalus durissus cascavella is an acid-soluble multisubunit glycoprotein of Mr 72,000 built up of two types of subunits, alpha and beta, linked by disulphide bonds. The mean apparent Mr of the reduced complex was around 12,000 by gel filtration under denaturating conditions. The Mrs of the alpha and beta subunits, with an apparent ratio of 1/1, were 12,600 and 13,580 by SDS-PAGE respectively. The Mr 72,000 glycoprotein is thought to be an alpha 3 beta 3 complex. The urea dissociated glycoprotein (Mr 72,000) retained its platelet-stimulating activity. It is concluded that the Mr 300,000 form isolated at acidic pH under native conditions, and showing a rosette - like, ring-shaped structure in the electron microscope as well as the Mr 144,000 form isolated at physiological pH under native conditions and active on platelets were the tetrameric and dimeric states of the molecule respectively.     

A new gene structure of the disintegrin family: a subunit of dimeric disintegrin has a short coding region

Disintegrin is a potent platelet aggregation inhibitor isolated from various snake venoms. The cDNA of the snake venom disintegrin family precursor is well-known to encode pre-peptide, metalloprotease, spacer, and disintegrin domains. Recently, new types of disintegrins, dimeric disintegrins, have been isolated, and their amino acid sequences were determined to be approximately 65 amino acid residues in each subunit. We isolated a novel heterodimeric disintegrin, acostatin, from the venom of Agkistrodon contortrix contortrix, which consisted of 63 and 64 amino acid residues in the alpha chain and beta chain, and both chains had the Arg-Gly-Asp (RGD) sequence for binding platelet GPIIb/IIIa. The cDNA lengths of the alpha chain and the beta chain of acostatin were 902 bp and 2031 bp, respectively. The acostatin alpha chain precursor, surprisingly, has the only disintegrin domain alone and lacked almost all of the pre-peptide and metalloprotease domains. The precursor of the acostatin beta chain belongs to a well-known motif of disintegrin precursors. Furthermore, both precursors of alpha and beta chains of another heterodimeric disintegrin, piscivostatin, also have the same domain structures as those of acostatin subunits. These results indicate that the cDNAs of heterodimeric disintegrin subunits have quite a different length of coding region and their precursors have a novel domain structure of disintegrin-family proteins.     

Molecular cloning and sequence analysis of aggretin, a collagen-like platelet aggregation inducer.

A cDNA library derived from the Malayan-pit-viper (Calloselasma rhodostoma) venom gland was constructed in the phagemid vector. Using the information of the N-terminal amino acid sequences of two subunits of aggretin, synthetic mixed-base oligonucleotides were employed as a screening probe for colony hybridization. Separate cDNA clones encoding for the alpha and beta chains of aggretin were isolated and sequenced. The results revealed that mature alpha and beta chains contain 136 and 123 amino acid residues, respectively. Aggretin subunits show high degrees of identity with respective subunits (50-60% for alpha, 49-58% for beta) of C-type lectin-like snake venoms. The identity to rattlesnake lectin is relatively lower (i.e., 39 and 30%). All cysteine residues in each chain of aggretin are well conserved and located at the positions corresponding to those of C-type lectins. Thus, three intracatenary disulfide bridges and an interchain disulfide bond between Cys83(alpha) and Cys75(beta) may be allocated. This is the first report regarding the entire sequence of venom GPIa/IIa agonist. According to the alignment of amino acid sequences, hypervariable regions among these C-type lectin-like proteins were revealed. These hypervariable regions are proposed to be the counterparts directly interacting with different receptors or different domains of a receptor on the surface of platelet.     

Rhodocetin, a novel platelet aggregation inhibitor from the venom of Calloselasma rhodostoma (Malayan pit viper): synergistic and noncovalent interaction between its subunits.

A novel platelet aggregation inhibitor, rhodocetin, was purified from the crude venom of Calloselasma rhodostoma. It inhibited collagen-induced platelet aggregation in a dose-dependent manner, with an IC50 of 41 nM. Rhodocetin has a heterodimeric structure with alpha and beta subunits, which could be separated on a nonreducing denaturing gel or reverse-phase HPLC column. Individually neither subunit inhibited platelet aggregation even at 2.0 microM concentration. Titration and reconstitution experiments showed that, when these subunits are mixed to give a 1:1 complex, most of its biological activity was recovered. The reconstituted complex inhibited platelet aggregation with an IC50 of 112 nM, about 3-fold less effective than the native molecule. Circular dichroism analysis revealed that the reconstituted complex had a spectrum similar to that of the native protein. By using surface plasmon resonance studies, we established that the stoichiometry of binding between the two subunits is 1:1 and the subunits interact with a Kd of 0.14 +/- 0.04 microM. The complete amino acid sequences of the alpha (15956.16 Da, 133 residues) and beta (15185.10 Da, 129 residues) subunits show a high degree of homology with each other (49%) and with the Ca2+-dependent lectin-related proteins (CLPs) (typically 29-48%) isolated from other snake venoms. Unlike the other members of the family in which the subunits are held together by an interchain disulfide bond, rhodocetin subunits are held together only through noncovalent interactions. The cysteinyl residues forming the intersubunit disulfide bridge in all other known CLPs are replaced by Ser-79 and Arg-75 in the alpha and beta subunits of rhodocetin, respectively. These studies support the noncovalent and synergistic interactions between the two subunits of rhodocetin. This is the first reported CLP dimer with such a novel heterodimeric structure.     

Purification and biochemical characterization of atroxase, a nonhemorrhagic fibrinolytic protease from western diamondback rattlesnake venom

Crotalus atrox venom contains a variety of proteases which render fibrinogen incoagulable and solubilize fibrin. One of these proteases was purified by using ion-exchange and gel permeation liquid chromatography. The protease, called atroxase, consists of a single nonglycosylated polypeptide chain with a molecular weight of 23,500 and an isoelectric point of pH 9.6. Amino acid analysis indicates atroxase to contain 206 residues with no sulfhydryl groups. Metal analysis found zinc and potassium at 1 mol/mol of protein, and calcium at 0.3 mol/mol of protein. Proteolytic activity is inhibited by ethylenediaminetetraacetate and alpha 2-macroglobulin. Maximal proteolytic activity occurs at pH 9.0 and 55 degrees C. Proteolytic specificity, using oxidized insulin B chain, is similar to that of several hemorrhagic toxins found within the same venom, yet atroxase shows no hemorrhagic activity and exhibits low lethality when tested on Swiss Webster mice. Atroxase, an A alpha, B beta fibrinogenase, cleaves the A alpha chain of fibrinogen first followed by the B beta chain and shows no effect on the gamma chain. The nonspecific action of the enzyme results in the extensive hydrolysis of fibrinogen which releases a variety of fibrinopeptides. Fibrin solubilization appears to occur primarily from the hydrolysis of alpha-polymer and unpolymerized alpha and beta chains. Although crude venom induces platelet aggregation, atroxase demonstrated no ability to induce or inhibit aggregation.     

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 μg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.     

Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 micromol. min(-1). mg(-1). The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP(+) oxidoreductase activity and catalyzed the reduction of NADP(+) with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content.     

一种新的烙铁头蛇毒肌肉毒素

用分子筛和快速蛋白质液相色谱从烙铁头（ＴＲｒｉｍｅｒｅｓｕｒｕｓ ｍｕｃｒｏｓｑｕａｍａｔｕｓ）蛇毒中分离了一个新的碱性肌肉毒素,命名为ＴＭＰＢ。它的分子量为１６０００,等电点为９．２．用蛋白质序列仪测定了其Ｎ端２４个氨基酸残基,ＴＭＰＢ与其他两个从同种蛇毒中分离到的碱性磷酯酶Ａ２的同源性分别为４１．７％和５４．２％《     

Purification, characterization, and cDNA cloning of a new fibrinogenlytic venom protein, Agkisacutacin, from Agkistrodon acutus venom

Agkisacutacin is a new fibrinogenlytic protein from Agkistrodon acutus venom. It consists of two heterologous subunits linked by an intersubunit disulfide bond. The cDNAs encoding the two chains of Agkisacutacin were cloned from a lambdagt11 cDNA library of the snake venom gland and sequenced, including the leader peptides (23/23 amino acid residues) and mature subunits (129/123 amino acid residues). It is structurally related to the family of IX/X-binding protein (IX/X-bp)-like proteins and shows high similarity (alpha-70%/beta-64%) to habu IX/X-bp from Trimeresurus flavoridis, but displays distinct biological activity with direct action on fibrinogen.     

Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin

A potent inhibitor of platelet aggregation and cell adhesion was isolated from the venom of Bothrops atrox. This peptide, referred to as batroxostatin, was composed of 71 amino acids and showed a high degree of homology with other snake venom peptides including trigramin, albolabrin, elegantin and applagin: all 12 cysteines and the RGD sequence (standard one-letter amino acid codes) aligned in the same position. Compared on a molar basis, the anti-platelet aggregation activity of batroxostatin was about 1000-times higher than that of RGDS. In addition, batroxostatin was about 400-times more potent than GRGDS at inhibiting melanoma cell adhesion to fibronectin. Batroxostatin covalently attached to plastic promoted adhesion of melanoma cells. The anti-GP140 antibody, recognizing beta 1 integrins, completely inhibited adhesion of mouse melanoma cells to batroxostatin. This observation, in addition to the inhibitory effect of batroxostatin on the adhesion of chick fibroblasts to fibronectin, suggests that batroxostatin interacts with integrins from both the beta 1 and beta 3 subfamilies.     

Amino acid sequence of a less-cytotoxic basic polypeptide (LCBP) isolated from the venom of the Indian cobra (Naja naja)

A less-cytotoxic polypeptide, designated as LCBP, was isolated from the venom of Naja naja by gel filtration on Sephadex G-50 followed by CM-cellulose chromatography. The cytotoxicity toward Yoshida sarcoma cells and lethal toxicity toward mice of LCBP were both one order of magnitude lower than that of cytotoxins and that of toxin A, respectively. LCBP is a single polypeptide consisting of 61 amino acid residues with four intramolecular disulfide linkages, and the amino acid sequence is the same as that of cardiotoxin-like basic polypeptide (CLBP) isolated from the venom of Naja naja atra. This is the first time that the same polypeptides were isolated from different cobra venoms.     

Amino acid sequence of a cardiotoxin-like basic polypeptide (CLBP) with low cytotoxic activity isolated from the venom of the Formosan cobra (Naja naja atra)

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.     

Molecular cloning of the penicillin G acylase gene from Arthrobacter viscosus.

Penicillin G acylase was purified from the cultured filtrate of Arthrobacter viscosus 8895GU and was found to consist of two distinct subunits with apparent molecular weights of 24,000 (alpha) and 60,000 (beta). The partial N-terminal amino acid sequences of the alpha and beta subunits were determined with a protein gas phase sequencer, and a 29-base oligonucleotide corresponding to the partial amino acid sequence of the alpha subunit was synthesized. An Escherichia coli transformant having the penicillin G acylase gene was isolated from an A. viscosus gene library by hybridization with the 29-base probe. The resulting positive clone was further screened by the Serratia marcescens overlay technique. E. coli carrying a plasmid designated pHYM-1 was found to produce penicillin G acylase in the cells. This plasmid had an 8.0-kilobase pair DNA fragment inserted in the EcoRI site of pACYC184.