Studies of an acidic cardiotoxin isolated from the venom of Mojave rattlesnake (Crotalus scutulatus).

A major lethal protein was isolated from the venom of Mojave rattlesnake (Crotalus scutulatus) by successive purification in DEAE column chromatography and isoelectric focusing. This homogeneous and monomeric form of toxin is designated as "Mojave toxin". Unlike basic neurotoxins or cytotoxins isolated from venoms of cobras, kraits and sea snakes, the Mojave toxin is an acidic protein with an isoelectric point of 4.7. The toxin is also different from crotoxin (from Crotalus durissus terrificus) which consists of both acidic and basic components. The molecular weight determined by Sephadex G-75 column chromatography resulted in a value of about 22 000. A singel protein band with a molecular weight of about 12 000, was observed after sodium dodecyl sulfate gel electrophoresis of the reduced Mojave toxin. Isoelectric focusing gel in the presence of 8 M urea also showed a single protein band, suggesting that the toxin is composed of subunits. Unlike the neurotoxic nature of the basic proteins from the venoms of Elapidae and sea snakes (Hydrophiidae) and crotoxin, Mojave toxin is cardiotoxic rather than neurotoxic. It is very likely that venoms of all rattlesnakes from North and Central America contain Mojave toxin as the common toxin.     

Sunflower seed protein: size and charge heterogeneity in subunits of the globulin fraction

The subunit heterogeneity of the globulin fraction of sunflower seeds was investigated by two dimensional electrophoresis, using isoelectric focusing in the first dimension and sodium dodecyl sulphate polyacrylamide gel electrophoresis in the second dimension. Under non reducing conditions, intermediary subunits B, C and D (molecular weight 54 000, 48 000 and 40 000, respectively) were focused within a pI range 5.4-6.0 but intermediary subunits A (molecular weight 60 000) focused within a pI range 6.3-6.8. Under reducing conditions the electrophoretic patterns show that intermediary subunits consist in large "acidic" and small "basic" subunits linked by disulphide bonds. The large subunits of B species are more acidic and less heterogeneous than the corresponding subunits of the A species. These results confirm that helianthinin had a "legumin-type" structure.     

RAPID CONCURRENT AUTOMATED FLUORIMETRIC ASSAY OF NORADRENALINE, DOPAMINE, 3, 4-DIHYDROXYPHENYLACETIC ACID, HOMOVANILLIC ACID AND 3-METHOXYTYRAMINE IN MILLIGRAM AMOUNTS OF NERVOUS TISSUE AFTER ISOLATION ON SEPHADEX G10

Abstract— Brain tubulin subunits were separated by a combination of isoelectric focusing and electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a two-dimensional polyacrylamide slab gel technique. Isoelectric focusing separated tubulin subunits into two major groups of bands, such that the more acidic group corresponded to the α subunit and the less acidic group corresponded to the β subunit. In addition, isoelectric focusing resolved the β subunit into two subspecies which differed slightly in isoelectric properties but were the same apparent molecular weight. The a subunit was resolved into many subspecies that appear to differ from each other by both apparent molecular weight and isoelectric properties.     

Physicochemical characterization of A and B subunits of Shiga toxin and reconstitution of holotoxin from isolated subunits

The A and B subunits of Shiga toxin were isolated by high performance liquid chromatography and their physicochemical properties were examined. The A subunit of Shiga toxin purified from culture supernatant was not nicked, but it could be nicked in vitro by trypsin. The isoelectric points of the A and B subunits were determined to be 8.2 and 5.8, respectively. Amino acid compositions of the two subunits were also determined. The isolated A and B subunits were reconstituted to form active holotoxin which showed lethal activity to mice which was similar to that of native Shiga toxin.     

Isoelectric-focusing properties and carbohydrate content of pea (Pisum sativum) legumin

Legumin from pea (Pisum sativum) is a molecule made up of six pairs of subunits, each pair consisting of an `acidic' subunit (mol.wt. about 40000) and a `basic' subunit (mol.wt. about 20000) linked by one or more disulphide bonds. The heterogeneity of legumin has been investigated by isoelectric focusing; undissociated legumin could not be focused satisfactorily, but legumin subunits could be analysed under dissociating conditions. 8m-Urea was not found to be a satisfactory medium for isoelectric focusing of legumin, as the `basic' subunits showed a shift in pI with time of incubation in urea. A new dissociating medium for isoelectric focusing, namely 50% (v/v) formamide, was used for analysis of legumin, which gave pI values of 5.0–5.3 for the `acidic' subunits, and 8.3–8.7 for the `basic' subunits. Both types of subunits were shown to be heterogeneous in charge and molecular weight by two-dimensional analysis employing isoelectric focusing in the first dimension and sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the second. The `basic' and `acidic' subunits of legumin were separated on the preparative scale by ion-exchange chromatography in 50% formamide. Carbohydrate attached to the protein was investigated as a possible cause of the heterogeneity of legumin subunits. However, both a fluorescent-labelling technique and a sensitive radioactive-labelling technique failed to show any carbohydrate bound to legumin subunits, and it was concluded that legumin is not a glycoprotein.     

Glycerophospholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exotoxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme.

An extracellular lethal toxin produced by Aeromonas salmonicida was purified by fast-protein liquid ion-exchange chromatography. The toxin is composed of glycerophospholipid:cholesterol acyltransferase (GCAT) (molecular mass, 25 kilodaltons) aggregated with lipopolysaccharide (LPS), the GCAT/LPS complex having a molecular mass of about 2,000 kilodaltons, estimated by gel filtration chromatography. The toxin is lethal for Atlantic salmon (Salmo salar L.) at a concentration of 0.045 micrograms of protein per g of body weight. The toxin is a hemolysin (T-lysin, active on fish erythrocytes), leukocytolysin, and cytotoxin. Antiserum to the purified toxin neutralized the lethal toxicity of the crude extracellular toxins, indicating this toxin to be the major lethal factor produced by A. salmonicida. In the crude extracellular products, small amounts of free GCAT were also present. This has been purified, and its activities and properties have been compared with those of the GCAT/LPS complex. The presence of LPS did not influence the GCAT activity of the enzyme with egg yolk or phosphatidylcholine (lecithin) as a substrate, but the specific hemolytic activity and lethal toxicity was about eightfold higher in the complexed form. Furthermore, the free GCAT was more susceptible to proteolytic and heat inactivation than was the GCAT/LPS complex. Recombination of LPS (phenol extracted from extracellular products of A. salmonicida) with free GCAT enhanced the hemolytic activity, lethal toxicity, and heat stability of the latter but did not influence its lecithinase activity. In native polyacrylamide gel electrophoresis, the GCAT/LPS complex and the recombined GCAT-LPS both showed a high-molecular-mass band which did not enter the gel, while the free GCAT produced a single band with low molecular mass. In isoelectric focusing gels, the GCAT/LPS and recombined GCAT-LPS produced a nonfocusing smear with pIs from pI 5.0 to 5.8, while the free GCAT produced a single band with pI 4.3. These data show that free GCAT can combine with LPS to produce a high-molecular-mass complex with enhanced toxicity and heat stability compared with those of free GCAT, similar to the preexisting GCAT/LPS complex, and indicate that the LPS moiety of the toxin plays an active role in toxicity.     

RAT BRAIN TUBULIN: SUBUNIT HETEROGENEITY AND PHOSPHORYLATION

Abstract— Evidence for multiple forms of the α and β subunits of tubulin isolated from rat brain has been obtained by means of SDS polyacrylamide gel electrophoresis, isoelectric focusing, and SDS hydroxylapatite column chromatography. Fourteen distinct bands, localized near pH 5.4, were formed when tubulin was subjected to isoelectric focusing in a gradient established with a very narrow range ampholyte mixture. Three tubulin subunits, a₁., α₂, and β, were separated by sodium dodecyl sulfate polyacrylamide slab gel electrophoresis in a second dimension. The β subunit was more acidic than the α subunits. Brain sections were incubated in tissue culture medium containing ³²P₁ and radiolabeled tubulin was subsequently isolated and subjected to electrophoresis. Only the β subunit was labeled. All radioactivity was associated with two or three adjacent bands on isoelectric focusing gels.     

An analysis of the subunit structure of the crystalloid protein complex from castor bean endosperm

Chromatographic and electrophoretic studies have shown that the subunits of the crystalloid protein, isolated from mature castor bean (Ricinus communis L. cv Hale) seed endosperm protein bodies, are heterogeneous with molecular weights in the range 49 to 53.5 kilodaltons (kD), and are quantitatively in unequal amounts. Each subunit comprises an αβ polypeptide pair which are reduced by 2-mercaptoethanol in two subgroups with molecular weights in the 29 to 34 kD and 20.5 to 23.5 kD ranges. Subunits and corresponding polypeptide pairs are also seen to be heterogeneous in pI following isoelectric focusing. In general, large polypeptides are acidic (pI 4.8-6.2) and small polypeptides basic (pI 7.4-9.4), although overlap of some isoelectric isomers does occur, notably in polypeptides derived from subunits which are quantitatively present in smaller amounts.     

An examination of the peroxidases from Lupinus albus L. hypocotyls

Peroxidases (EC 1.11.1.7) from hypocotyls of Lupinus albus L. cv. Rio Maior have been characterised using one- and two-dimensional, native electrophoretic techniques. Data are presented showing the complexity in charge and molecular size or shape of these peroxidases. We report the finding of a new acidic peroxidase and several new basic peroxidases in these hypocotyls, and of their stability to treatments considered to break ligand-induced variants and conformational variants derived from differences in polypeptide folding. Densitometric data demonstrate that these new peroxidases contribute up to 60 of the total peroxidase activity in hypocotyls. Studies of intercellular fluid, cell-wall and soluble fractions, with assays of purity were conducted in an attempt to define the subcellular locations of these additional peroxidases. The acidic form (pI 4.1) is greatly enriched in soluble fractions, three of the basic peroxidases (pIs 9.5, 9.7 and >9.7) are strongly associated to the cell wall, ad a minor, basic component (pI 9.7) is enriched in the intercellular fluid. Individual peroxidase activities with the substrates coniferyl alcohol, ferulic acid or indole acetic acid were compared by densitometric analysis of zymograms with those for guaiacol, and notable differences between these peroxidases in their capacity to oxidise indole acetic acid in vitro were identified. The possible functions of these peroxidases in vivo and their implications to current understanding of peroxidases in L. albus are discussed.Abbreviations APAGE anionic polyacrylamide gel electrophoresis - CA coniferyl alcohol - CPAGE cationic polyacrylamide gel electrophoresis - IEF isoelectric focusin - NEIEF non-equilibrated isoelectric focusing - 2D two dimensional - pI isoelectric point - RCPAGE reversed current polyacrylamide gel electrophoresis     

Characterization of the acidic lectins from winged bean seed (Psophocarpus tetragonolobus(L.)DC)

The seeds of winged bean, Psophocarpus tetragonolobus(L.)DC, contain two distinct groups of lectins characterized by different erythrocyte hemagglutinating specificities and isoelectric points. Three acidic lectins (I, II, and III) (pI approximately 5.5) were purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP-Sephadex C-25. These lectins are glycoproteins with relative molecular mass of 54,000. The total carbohydrate content of the acidic lectins was 7% and was comprised of mannose, N-acetylglucosamine, fucose, and xylose in amounts corresponding to 9.2, 4.8, 1.6, and 7.0 mol/54,000 g, respectively. Electrophoresis in dodecyl sulfate, in the presence and absence of 2-mercaptoethanol, gave a single subunit of apparent relative molecular mass 30-32,000, somewhat higher than expected from the native relative molecular mass. On isoelectric focusing in 8 M urea the subunits of the acidic lectins did not show any significant charge heterogeneity as found for the winged bean basic lectins. The acidic lectins have very similar amino acid compositions. They contain essentially no half-cystine, 1-2 methionine residues, and are rich in acidic and hydroxy amino acids. The amino-terminal sequences of lectins II and III were identical while the amino-terminal sequence of lectin I contained five differences in the first 25 residues; the acidic lectins showed extensive sequence homology with the winged bean basic lectins, the other one-chain subunit lectins and the beta subunit of the two-chain subunit legume lectins. The acidic lectins agglutinated trypsinized human (type A, B, AB, and O) erythrocytes but not trypsinized rabbit erythrocytes. They were inhibited by various D-galactose derivatives and D-galactose-containing disaccharides and trisaccharides. N-Acetylgalactosamine was the best inhibitor, and the specificity appears to be directed to beta-D-galactosides. However, compared with winged bean basic lectins and soybean lectin, the winged bean acidic lectins show a low affinity for the inhibitory sugars.     

Charge heterogeneity of cholera toxin and its subunits

Abstract Analytical isoelectric focusing (IEF) in thin layers of polyacrylamide gels resolved cholera toxin into 3 isomeric forms differing in charge (isoelectric points 6.80, 6.65 and 6.55). All these forms had identical molecular weights, and were also antigenically similar, as demonstrated by their reactivity to antisera to cholera toxin. Both the B and A subunits possessed charge heterogeneity. The B subunit was detected in a free form when a solution of cholera toxin was aged for a few days. Antisera to cholera toxin, irrespective of mode of immunisation, contained antibodies to both the intact cholera toxin and the free B subunit as demonstrated by the immunoblotting technique based on IEF.     

Characterization of ferritin from human placenta. Implications for analysis of tissue specificity and microheterogeneity of ferritins

Mammalian ferritins can be resolved into multiple components by isoelectric focusing, and each tissue contains a characteristic subset of isoferritins. Ferritin isolated from human liver was compared to acidic ferritin isolated from mid-gestational human placenta to define a structural basis for ferritin heterogeneity. Placenta ferritin contained several major bands with isoelectric points in the range of pI = 4.7-5.0 which were more acidic than the predominant isoferritins of human liver. Ferritin from each tissue was resistant to denaturation by 10 M urea and appeared to be identical by electron microscopy. Circular dichroism measurements revealed that placenta ferritin had substantially less ordered secondary structure than liver ferritin. Both types of ferritin contained only two subunits when analyzed by electrophoresis in sodium dodecyl sulfate gels, but isoelectric focusing of dissociated subunits in urea revealed 6-7 different components. In this system, placenta ferritin was enriched in the more acidic subunits and it completely lacked the most basic subunits noted in liver ferritin; placental ferritin had no unique components. Differences in isoelectric points among assembled ferritins from these two tissues appear to result from different proportions of these acidic and basic subunits.     

Isoelectric focusing patterns of staphylococcal exfoliative toxin

Multiple components in two antigenic variants of staphylococcal exfoliative toxin were demonstrated by isoelectric focusing in polyacrylamide gel. The main components had an isoionic point of pH=7.0 while other components ranged from pI=4.5 to pI=8.5. Each individual component was shown to possess full biologic and serologic activity after focusing. Isoelectric focusing in the presence of 8 M urea also showed microheterogeneity in exfoliative toxin preparations. The various components appear to be reversible conformers, possibly raised by exposure to the ph gradient of the ampholines.     

Purification of an extracellular thiol-dependent hemolysin from Bacillus alvei]

Alveolysin a sulfhydryl-dependent cytolytic extracellular protein released by Bacillus alvei has been purified by salting-out by ammonium sulfate, gel filtration, isoelectric focusing on pH gradient and chromatography on DEAE-cellulose. The purified protein after reduction by thiols (active hemolytic form) proved homogeneous by disc polyacrylamide gel electrophoresis and by gel immunodiffusion. The molecular weight was 60,000 daltons, Two molecular forms of pI 5.1 and 7.0 were detected by gel isoelectrofocusing. The toxin was lethal to the mouse. Lytic activity was inhibited by cholesterol and antistreptolysin O anstisera. Immunological cross-reaction was observed between alveolysin and streptolysin O.     

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.     

Purification of RNA-core induced streptolysin S, and isolation and haemolytic characteristics of the carrier-free toxin

RNA-core (RNAase-resistant fraction of yeast RNA) induced streptolysin S (SLS) was purified (40% recovery) to apparent electrophoretic homogeneity by hydroxylapatite chromatography followed by gel filtration on Sephadex G-100 in the presence of 6 M-guanidine. HCl. The specific activity of the purified toxin was 3 X 10(6) haemolytic units (mg protein)-1. The Mr of the toxin was below 4000 on the basis of SDS-PAGE and 20 000 by gel filtration in guanidine. HCl. High-voltage isoelectric focusing of the purified toxin allowed the isolation of the carrier-free SLS peptide for the first time. This peptide was basic (pI 9.2) as compared to native SLS (pI 3.6). The native toxin and the peptide had similar haemolytic properties except for the high lability of the peptide, which was stabilized by RNA-core. The Mr of the denatured peptide was about 1800, as estimated by gel filtration.     

Comparative study of three phospholipase A2s from the venom of Vipera aspis

1. Three phospholipase A2s, PLA2-I, PLA2-II and PLA2-III, were isolated from Vipera aspis venom by gel filtration and ion exchange chromatography. 2. Purified PLA2-I, -II and -III have mol. wts of 30,200, 16,000 and 13,500, and isoelectric points of 9.45, 7.65 and less than 4.1, respectively. 3. PLA2-I consists of an acidic subunit (mol. wt 13,700, pI: less than 3.5) and a basic subunit (mol. wt 16,500, pI: 10.6), which can be separated under highly acidic conditions. 4. PLA2-I possessed lethal activity and LD50 for this preparation was estimated to be 0.288 (0.209-0.397) micrograms/g, while lethality was not observed when PLA2-II, -III or each subunit of PLA2-I were administered. 5. Capillary permeability-increasing activity was found in the samples which possessed basic isoelectric points. Additionally, PLA2-I and its basic subunit drastically prolonged activated partial thromboplastin time of platelet rich plasma. 6. Intramuscular injections of PLA2-I, -II and -III increased serum creatine phosphokinase activity in mice, indicating that damage in muscle was caused by these enzymes. 7. NH2-terminal sequences of the three PLA2s were compared with other phospholipase A2s from snake venoms. Furthermore, antigenicities were tested using antiserum prepared against each sample.     

Purification and physicochemical properties of a unique Vero cell cytotoxin from Escherichia coli O157:H7

A unique Vero cell cytotoxin has been purified to homogeneity from a strain of Escherichia coli O157:H7, using ultrafiltration with Pellicon membrane cassettes and chromatography with QAE Sephadex A-50. SDS-PAGE showed the molecular weight of the toxin to be 64,000 and the absence of subunits. Based on analytical isoelectric focusing, the toxin had pI of 5.2. This Vero cell toxin was lethal to mice and showed pathological abnormalities of the mouse colonic mucosa when administered intraperitoneally. Vero cell cytotoxicity of this toxin was not neutralizable with rabbit antiserum to Shiga toxin. Based on physicochemical and immunological properties, this toxin is different from the Shiga-like toxin previously found in this organism.     

Formation of Pseudo- and Hybrid-11S Globulins from Subunits of Soybean and Broad Bean 11S Globulins

Pseudo- and hybrid-11S globulins were reconstituted from native acidic and basic subunits of soybean and broad bean 11S globulins. The subunit structures of these two globulins are known to be similar to each other. Pseudo-11S globulins were formed in combinations between glycinin acidic subunit (G-AS_{1 + 2}) and glycinin basic subunit (G-BS) and between legumin acidic subunit (L-ASII) and legumin basic subunit (L-BS). Hybrid-11S globulins were formed in combinations between G-AS_{1 + 2} and L-BS and between L-ASII and G-BS. The yields of the reconstituted 11S components of G-AS_{1 +2} + G-BS and G-AS_{1 + 2} + L-BS were lower than those of L-ASII + G-BS and L-ASII + L-BS. These pseudo- and hybrid-11S globulins were similar to native 11S globulins; they all consisted of reconstituted intermediary subunits which were composed of acidic and basic subunits linked by disufide bridges and had molecular weights similar to those of native 11S globulins. However, the dissociation-association behaviors of pseudo-glycinin and hybrid-11S globulins were different from those of native 11S globulins.