Purification and characterization of alpha-toxin of Clostridium oedematiens type A

The alpha-toxin of Clostridium oedematiens type A was purified from culture filtrate by two steps of column chromatography and repeated gel filtration. The purified alpha-toxin proved homogeneous in polyacrylamide gel electrophoresis and agar gel double diffusion. The molecular weight of the alpha-toxin was estimated at 280,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and at 260,000 by gel filtration on a Sephadex G-200 column. The isoelectric point determined by isoelectric focusing polyacrylamide gel electrophoresis was 6.1. No dissociation of the purified alpha-toxin into subunits was demonstrated in sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 50% lethal and edematizing doses per mg protein of the purified alpha-toxin were 5.9 X 10(4) and 5.9 X 10(5), respectively. The L +/50 doses per mg protein of the toxin was 4.6 X 10(3). The purified alpha-toxin, when injected intradermally into the rabbit skin, induced increased vascular permeability. The toxin contained little or no hemolytic or lecithinase activity. These results attest that the lethal, edematizing and vascular permeability-enhancing activities elicited by C. oedematiens type A culture reside on the same protein molecule.     

Orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor. Fluorescence studies

Four flourescein isothiocyanate (FITC) derivatives of Naja naja siamemsis 3 neurotoxin (alpha-toxin), labeled at the epsilon-amino groups of Lys-23, Lys-35, Lys-49, or Lys-69, and a tetramethylrhodamine isothiocyanate (TRITC) derivative, labeled at epsilon-amino group of Lys-23, were prepared and used to analyze the orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor (AcChR) relative to both the plane of the membrane and the central ion channel. Fluorescence-quenching studies of the AcChR-bound FITC derivatives indicated significant solute accessibility to each site of labeling and suggested that none of the sites of FITC labeling is included in the binding surface of the alpha-toxin. Labeling of Lys-23 with TRITC did not affect the affinity of the alpha-toxin toward the AcChR and confirmed, contrary to some previous reports, a minimal role of Lys-23 in the binding surface of the alpha-toxin. Measurements of energy transfer between the lipid-membrane surface and the sites of labeling on receptor-bound alpha-toxin derivatives show that the relative distances of closest approach between the surface of the lipid membrane domain and the sites of labeling are in the order Lys-23 less than or equal to Lys-49 less than Lys-35 less than or equal to Lys-69. Energy transfer between AcChR tryptophans and the sites of labeling of bound derivatives was about 50% greater to Lys-49 than to Lys-23, Lys-35, or Lys-69, suggesting that Lys-49 is closer to receptor tryptophans and to the center of the extracellular domain of the receptor than Lys-23, Lys-35, or Lys-69. Combined with previous observations that the tip of the central loop of the alpha-toxin directly interacts with the AcChR, the above results suggest a model of the approximate orientation of the snake neurotoxins on the receptor. This model shows the tip of the central loop of the toxin directly interacting with the receptor surface and the major axis of the neurotoxin tilting from a perpendicular projection from the membrane. The surface of the alpha-toxin that includes Lys-23 projects away from the central ion channel and the surface that includes Lys-35 and Lys-69 faces the ion channel.     

Fluorescence energy transfer between cobra alpha-toxin molecules bound to the acetylcholine receptor

An approach was developed with steady state fluorescence energy transfer measurements to examine the spatial relationship between the two alpha-toxins bound to the acetylcholine receptor. By taking advantage of the slow dissociation rates of alpha-toxins (Naja naja siamensis 3) from the receptor and of the equal probability with which alpha-toxins bind to the two alpha-toxin-binding sites, we derived an equation which allows prediction of a "true" efficiency of transfer based on the relationship between fractional site occupancy and the observed transfer efficiency ascertained from donor quenching. Using this approach, we examined the efficiency of energy transfer between two fluorescently labeled alpha-toxins, N epsilon-fluorescein isothiocyanate lysine 23 alpha-toxin and monolabeled tetramethylrhodamine isothiocyanate alpha-toxin bound to the receptor from the Torpedo californica electric organ. Significantly greater (32 versus 14%) energy transfer was observed with the membrane-associated than with the solubilized receptor, suggesting that transfer between fluorophores on separate receptor molecules is greater than that occurring intramolecularly between the two sites on the receptor. The magnitude of the distances calculated from the intrareceptor energy transfer efficiency combined with the considerable inter-receptor energy transfer indicate that the fluorophores would reside on the outer perimeter of the receptor molecule rather than near the central axis perpendicular to the plane of the membrane.     

Purification and characterization of a neurotoxin from the venom of Ophiophagus hannah (king cobra)

A neurotoxin, Oh9-1, from the venom of Ophiophagus hannah was isolated by a combination of ion-exchange chromatography and reverse phase HPLC. Amino acid sequence analysis revealed that Oh9-1 consists of 57 amino acids and eight cysteine residues. This protein was mainly constituted with beta-sheet as evidenced by CD spectrum. Oh9-1 inhibited carbachol-induced muscle contraction in an irreversible manner and the dose for achieving 50% inhibition was approximately fourfold that of alpha-bungarotoxin. Since the residues in alpha-neurotoxins closely involve in the binding with acetylcholine receptors are not highly conserved in this toxin molecule, Oh9-1 represents a novel type of neurotoxin structurally distinct from alpha-neurotoxins.     

Purification and characterization of four different alkaline phosphatases from Spirographis spallanzanii

Alkaline phosphatases (APs) present in Spirographis spallanzanii were extracted by water homogenization of butanol treatment. The enzymes solubilized as above were purified by acetone fractionation and then by DEAE-cellulose and Sephadex G-200 chromatography; separation of four AP forms was achieved, which were characterized by studying their molecular and catalytic properties. The various APs differ in molecular weight, electrophoretic mobility, optimum pH, but show similar Km values and substrate inhibition pattern. Kinetic studies carried out with several inhibitors show, in particular, the existence in these APs of a second binding site and suggest a possible role for them in the metabolism of phosphoric esters of the sugars.     

Purification and partial characterization of four proteins from human parotid saliva

Four proteins, which have been designated A, B, C and D, have been purified from human parotid saliva. These proteins are the major constituents of parotid saliva which migrate rapidly to the anode in polyacrylamide electrophoresis at pH9.5. Gel filtration and polyacrylamide electrophoresis were employed in the purification procedures. After purification all four preparations were tested for homogeneity by electrophoresis at pH2.8 and 9.5, by isoelectric focusing in the pH range 3-10, by immunodiffusion, and by sedimentation in the analytical ultracentrifuge. None of the proteins showed significant activity in assays for amylase, acid and alkaline phosphatase, protease, lysozyme, ribonuclease, peroxidase, beta-glucuronidase, beta-galactosidase, iron-binding activity and esterase. No cross-reactions were detected with antisera specific for lactoferrin and 15 serum proteins. All four proteins were rich in glutamic acid, proline and glycine and were lacking completely the sulphur-containing amino acids. Proteins A and C contained no threonine or tyrosine. Carbohydrate could be demonstrated only in protein A at a concentration of 4% of the total protein.     

Purification and characterization of a novel phospholipase A2 from king cobra (Ophiophagus hannah) venom

A novel phospholipase A₂, designated as Oh-DE-2, was isolated from the venom ofOphiophagus hannah (king cobra) by successive chromatography on SP-Sephadex C-25, DE-52, and Q-Sepharose columns. Oh-DE-2 with pI 5.1 showed an apparent molecular weight of 14 kD as revealed by SDS-PAGE and gel filtration. The amino acid sequence was homologous with those of PLA₂s from Elapidae venoms. Oh-DE-2 was effectively inactivated byp-bromophenacyl bromide, indicating that the conserved His-48 is essential for its enzymatic activity. However, modification of the conserved Trp-19 did not cause a precipitous drop in the enzymatic activity of Oh-DE-2 as observed with PLA₂s fromNaja naja atra andBungarus multicinctus venoms. A quenching study showed that the microenvironment of Trp in Oh-DE-2 was inaccessible to acrylamide, iodide, or cesium, a finding which was different from those observed with PLA₂s fromN. naja atra andB. multicinctus venoms. These results might suggest that, unlike other PLA₂ enzymes, Trp-19 in Oh-DE-2 is not directly involved in its enzymatic mechanisms.     

Clostridium perfringens alpha-toxin: characterization and mode of action

Clostridium perfringens type A strains that produce alpha-toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production. Intramuscular injection of the toxin or Bacillus subtilis carrying the alpha-toxin gene causes myonecrosis and produces histopathological features of the disease. Immunization of mice with alpha-toxin or fragments of the toxin prevents gas gangrene caused by C. perfringens. The toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis. These biological activities are closely related to PLC and/or SMase activities. However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of alpha-toxin. Based on the isolation and characterization of the gene for alpha-toxin and a comparison of the toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of alpha-toxin and the mode of action of the toxin. To provide a better understanding of the role of alpha-toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of alpha-toxin.     

Purification and characterization of anticomplement factor (cobra venom factor) from the Naja naja atra venom

Anticomplement factor (cobra venom factor) from the venom of Naja naja atra was purified by means of successive chromatography on DEAE-cellulose, Sephadex G-200 and Sepharose CL-6B. The purified anticomplement factor was homogeneous as judged by polyacrylamide discontinuous gel electrophoresis at pH 9.4. The yield from 3.0 g of the crude venom was approx. 28 mg. The molecular weight was estimated to be about 156 000 by SDS-polyacrylamide gel electrophoresis. The isoelectric point was about 5.2. SDS-polyacrylamide gel electrophoresis of the anticomplement factor in the presence of dithiothreitol demonstrated that the molecule possesses three different polypeptide chains cross-linked covalently to one another by disulfide bridge(s). By SDS-polyacrylamide gel electrophoresis, the molecular weight of each subunit was determined to be approx. 77000, 47500 and 29 000, respectively. All subunits were stained with Coomassie brilliant blue G-250 and periodate-Schiff reagent, indicating these subunits to be glycoprotein. Distribution of the anticomplement factor in various snake venoms, which shows cross-reactivity against the anti-Naja naja atra anticomplement factor antiserum, was examined. From the results, all venoms belonging to cobra family in the Elapidae tested so far were found to contain such cross-reactivity.     

Purification, characterization, and partial amino acid sequence of nerve growth factor from cobra venom.

The nerve growth factor (NGF) from Naja naja (cobra) venom has been purified and its structure compared to the NGF from mouse submaxillary gland. A two-step purification procedure has been devised, consisting of a gel filtration step in 1 M acetic acid followed by chromatography of the active pool on carboxymethylcellulose at pH 5. The molecular weight of the native protein was found to be 28000, and this value was reduced by approximately one-half under denaturing conditions. These values are comparable to those obtained for mouse 2.5S or betaNGF. Tryptic peptide maps of S-[14C]carboxymethyl NGF gave the number of labeled peptides expected for a structure composed of two identical or very similar subunits. Thus, the quaternary structures of mouse and cobra NGF are the same. Cyanogen bromide (CNBr) treatment of Naja naja NGF produced three fragments, of which two were purified to homogeneity. These fragments and the whole protein were analyzed in the automated protein Sequencer. The amino-terminal CNBr fragment of the protein was also subjected to digestion by thermolysin and the resultant peptides were purified and characterized. These data plus those from the characterization of the tryptic peptides provided the basis of the construction of a tentative primary structure of Naja naja NGF which is approximately 60% identical with mouse NGF.     

Purification and characterization of four isolectins of mushroom (Agaricus bisporus)

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.     

Purification of alpha-toxin from Staphylococcus aureus and application to cell permeabilization

Crude alpha-toxin was produced by Staphylococcus aureus, strain Wood 46. The amount of exotoxin was monitored during growth and all subsequent purification steps by determination of its hemolytic activity against rabbit erythrocytes. The culture supernatant was treated with ammonium sulfate (75% saturation). The resulting precipitate was dialyzed and subjected to cation-exchange chromatography. The fractions containing the hemolytic activity were further purified by gel chromatography. The final product was enriched by a factor of 8.5 compared to the crude toxin. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis the purified toxin exhibited one major band. It caused the release of 86Rb+ and ATP from rat insulinoma (RIN A2) as well as pheochromocytoma cells (PC12) in culture, indicating efficient permeabilization of their plasma membranes for small molecules.     

Purification and biochemical characterization of four iron superoxide dismutases in Trypanosoma cruzi

Four superoxide dismutase (SOD) activities (SOD I, II, III, and IV) have been characterized in the epimastigote form of Trypanosoma cruzi. The total extract was subjected to two successive ammonium sulphate additions between 35 and 85%, and the resulting fraction was purified using two continuous chromatography processes (ion exchange and filtration). Enzymes were insensitive to cyanide but sensitive to hydrogen peroxide, properties characteristic of iron-containing SODs. The molecular masses of the different SODs were 20 kDa (SOD I), 60 kDa (SOD II), 50 kDa (SOD III) and 25 kDa (SOD IV), whereas the isoelectric points were 6.9, 6.8, 5.2 and 3.8, respectively. Subcellular location and digitonin experiments have shown that these SODs are mainly cytosolic, with small amounts in the low-mass organelles (SOD II and SOD I) and the mitochondrion (SOD III), where these enzymes play an important role in minimizing oxidative damage.     

Purification and characterization of two acidic phospholipase A2 enzymes from king cobra (Ophiophagus hannah) snake venom

1. The two major phospholipase A2 enzymes (OHPLA-DE1 and OHPLA-DE2) of king cobra (Ophiophagus hannah) venom have been purified to electrophoretic homogeneity. 2. The isoelectric points of OHPLA-DE1 and OHPLA-DE2 were 3.81 and 3.89, respectively and the Mws were 14,000 and 15,000, respectively, as estimated by Sephadex G-75 gel filtration chromatography; and 14,000 as estimated by SDS-PAGE. 3. The enzymes were not lethal to mice at a dosage of 10 micrograms/g body wt by i.v. route. Both phospholipase A2 enzymes, however, exhibited moderate edema-inducing and anti-coagulant activities. 4. Bromophenacylation of the enzymes reduced the enzymatic activity drastically but did not affect the edema-inducing activity of the enzymes.     

Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.