Purification and some properties of cytotoxin produced by Clostridium difficile

The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q. The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol. Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively. Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000. It had an isoelectric point of 6.6. The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10. The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng. It was also demonstrated that the toxin is antigenically different from enterotoxin of C. difficile.     

Snake venom toxins. The amino-acid sequences of three toxins (CM-8, CM-11 and CM-13a) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-8, CM-11, and CM-13a) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex G-50 and by ion-exchange chromatography on CM-cellulose. Whereas toxin CM-8 and CM-11 comprise 60 amino acid residues, toxin CM-13a contains 61 residues. All three toxins are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of these toxins have been elucidated. The reduced and S-carboxymethylated toxins were digested with trypsin and chymotrypsin and the peptides purified by ion-exchange chromatography, gel filtration and chromatography or electrophoresis on paper. The Edman procedure, either through the use of the automatic sequencer or by manual manipulation, was employed to obtain the sequence of the intact toxins and the pure peptides. The chymotryptic digests provided the necessary overlapping peptides which allowed the alignment of the tryptic peptides. The properties of the three toxins were compared with those of the cytotoxin group. The toxicities the serological properties, the sequences and the invariant amino acid residues of toxin CM-8 and CM-11 resemble the corresponding properties of the cytotoxin group. The sequence and serological properties of toxin CM-13a show that it is related to the cytotoxin group, but its toxicity is much lower than those encountered in the cytotoxin group.     

Amino acid sequence of cytotoxin IIa isolated from the venom of the Indian cobra (Naja naja)

A cytotoxic basic polypeptide, designated as cytotoxin IIa, was purified to homogeneous state from the venom of the Indian cobra (Naja naja) by a combination of gel filtration on Sephadex G-50, CM-cellulose chromatography, and fast protein liquid chromatography. Cytotoxin IIa is a single polypeptide consisting of 60 amino acid residues with four intramolecular disulfide linkages. The toxin showed high cytotoxicity toward Yoshida sarcoma and ascites hepatoma cells as did cytotoxins I and II isolated from the same venom. Analysis of the amino acid sequence revealed that cytotoxin I, IIa, and II are highly homologous in their primary structures and that cytotoxin IIa differs from cytotoxin I only in having Phe 25 and Val 52 in place of Tyr 25 and Glu 52 residues.     

Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Snake venom toxins. The amino acid sequence of three toxins (CM-2e, CM-4a and CM-) from Naja haje annulifera (Egyptian cobra) venom.

Three toxins (CM-2e, CM-4a and CM-7) were purified from the venom of Naja haje annulifera by gel filtration on Sephadex and by ion-exchange chromatography on CM-cellulose. They comprise 60 amino acid residues and are cross-linked by four intrachain disulphide bridges. The complete amino acid sequences of the three toxins have been elucidated. The toxicities, the serological properties, the sequences and the invariant amino acid residues of toxin CM-2e, CM-4a and CM-7 resemble the corresponding properties of the cytotoxin group.     

Snake venoms. The amino-acid sequence of protein S2C4 from Dendroaspis jamesoni kaimosae (Jameson's mamba) venom.

A major component (S2C4) was purified from Jameson's mamba by gel filtration on Sephadex G-50 and ion-exchange chromatography on CM-cellulose. Protein S2C4 comprises 62 amino acid residues including 8 half-cystine residues. The complete amino acid sequence of the protein has been established. The sequence and the invariant amino acid residues of protein S2C4 resemble a short neurotoxin, a long neurotoxin, a cytotoxin and an angusticeps type protein. However, the position of its four disulphide bridges differs from those encountered in a short neurotoxin or a cytotoxin. Mixtures of protein S2C4 and angusticeps type proteins revealed a marked synergistic effect, in that their toxicity in combination was greater than the sum of their individual toxicities.     

Snake venom toxins--II. The primary structures of cytotoxin homologues S3C2 and S4C8 from Aspidelaps scutatus (shield or shield-nose snake) venom

1. Cytotoxin homologues S3C2 and S4C8 from Aspidelaps scutatus were purified by gel filtration and ion exchange chromatography. 2. They consist of 63 amino acids including eight half-cystines. The toxicities of S3C2 and S4C8 were determined and LD50 values of 6.6 and 9.4 micrograms/g mouse were, respectively, found. 3. The complete primary structures of toxins S3C2 and S4C8 have been determined. The two toxins resemble the cytotoxin type toxins and in the cytotoxin homologues the ten structurally invariant amino acids of the neurotoxins and the cytotoxins are conserved.     

Purification and some properties of low-molecular-weight proteins of Aspidelaps scutatus (shield or shield-nose snake) venom

1. Fourteen low-molecular-weight proteins from Aspidelaps scutatus venom were purified by gel filtration and ion exchange chromatography. 2. The proteins (toxins) have i.v. LD50 values ranging from 0.12 to 54 micrograms/g mouse. 3. The amino-terminal sequences of the proteins show that 12 of the 14 proteins were pure. 4. The proteins were preliminarily assigned as phospholipases A2, long neurotoxins and cytotoxin homologues.     

Adherence of Vancomycin to Proteins

Vancomycin possesses the unusual property of promoting the aggregation of proteins. It also binds to itself (dimerization). Both properties may be related to its antimicrobial activity and we report here procedures to measure them. The position of the negative ellipticity band in the near ultraviolet circular dichroism spectrum of the vancomycin monomer shifts as a function of antibiotic concentration and can be used to readily determine the monomer-dimer equilibrium constant. These measurements complement those performed by high-resolution gel filtration to measure the same process. Aggregation of purified proteins was determined by turbidity measurements. Both dimerization and protein aggregation are influenced by anions whose effectiveness is related to their carboxyl pK_a values, thus linking these two properties.     

N-terminal PDZ domain is required for NHERF dimerization

NHERF, a 55 kDa PDZ-containing protein, binds receptors and ion transporters to mediate signal transduction at the plasma membrane. Recombinant NHERF demonstrated an apparent size of 150 kDa on gel filtration, which could be reduced to approximately 55 kDa by protein denaturing agents, consistent with the formation of NHERF dimers. Biosensor studies established the time- and concentration-dependent dimerization of NHERF. Overlays of recombinant NHERF fragments suggested that NHERF dimerization was principally mediated by the N-terminal PDZ-I domain. In PS120 cells, reversible protein phosphorylation modulated NHERF dimerization and suggested a role for NHERF dimers in hormonal signaling.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein.

Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2. We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer. The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography. The A2 protein's identity was confirmed by two-dimensional gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide. Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide. The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer.     

Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine

Arabidopsis thaliana threonine deaminase (TD) is a tetramer composed of identical approximately 59600 Da subunits. TD activity has been shown to be inhibited by isoleucine. This effect is reversed by a large excess of valine. Nondenaturant gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry experiments demonstrated that binding of isoleucine on TD induces dimerization of the enzyme, whereas tetramerization is restored by addition of a high valine concentration. Nondenaturant gel filtration and electrospray ionization mass spectrometry of the enzyme in the presence of increasing amounts of isoleucine suggest a fast equilibrium between the tetramer and the dimer. Finally, study of TD mutants allowed us to focus on the specific role of each isoleucine-binding site.     

Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

Apple four in human blood coagulation factor XI mediates dimer formation.

Human blood coagulation factor XI is a dimer composed of two identical subunits. Each subunit contains four apple domains as tandem repeats followed by a serine protease region. A disulfide bridge between Cys321 of each fourth apple domain links the subunits together. The role of Cys321 in the dimerization of factor XI was examined by mutagenesis followed by expression of its cDNA in baby hamster kidney cells. The recombinant proteins were then purified from the tissue culture medium and shown to have full biological activity. Normal recombinant factor XI was secreted as a dimer as determined by SDS-PAGE, while recombinant factor XI-Cys321 Ser migrated as a monomer under these conditions. Gel filtration studies, however, revealed that each protein existed as a dimer under native conditions, indicating that the disulfide bond between Cys321 of each factor XI monomer was not necessary for dimer formation. The fourth apple domain (apple4) of factor XI was then introduced into tissue plasminogen activator (tPA) to investigate its role in the dimerization of other polypeptide chains. The fusion protein, containing apple4 (apple4-tPA), formed dimers as detected by SDS-PAGE and gel filtration. Furthermore, dimerization was specific to apple4, while apple3 had no effect on dimerization. These data further indicated that the apple4 domain of factor XI mediates dimerization of the two subunits and the interchain disulfide bond involving Cys321 was not essential for dimer formation.     

Correlation between catalytic activity and monomer-dimer equilibrium of bacterial alanine racemases

From the reaction mechanism and crystal structure analysis, a bacterial alanine racemase is believed to work as a homodimer with a substrate, l-alanine or d-alanine. We analysed oligomerization states of seven alanine racemases, biosynthetic and catabolic, from Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, P. putida and P. fluorescens, with three different methods, gel filtration chromatography, native PAGE and analytical ultracentrifugation. All alanine racemases were proved to be in a dynamic equilibrium between monomeric and dimeric form with every methods used in this study. In both biosynthetic and catabolic alanine racemases, association constants for dimerization were high for the enzymes with high V(max) values. The enzymes with low V(max) values gave the low association constants. We proposed that alanine racemases are classified into two types; the enzymes with low and high-equilibrium association constants for dimerization.     

Structure of DNA polymerase delta from Saccharomyces cerevisiae

DNA polymerase delta (Pol delta) from Saccharomyces cerevisiae consists of three subunits, Pol3 (125 kDa), Pol31 (55 kDa), and Pol32 (40 kDa), present at a 1:1:1 stoichiometry in purified preparations. Previously, based on gel filtration studies of Pol delta, we suggested that the enzyme may be a dimer of catalytic cores, with dimerization mediated by the Pol32 subunit (Burgers, P. M., and Gerik, K. J. (1998) J. Biol. Chem. 273, 19756-19762). We now report on extensive gel filtration, glycerol gradient sedimentation, and analytical equilibrium centrifugation studies of Pol delta and of several subassemblies of Pol delta. The hydrodynamic parameters of these assemblies indicate that (i) Pol32 is a rod-shaped protein with a frictional ratio f/f(0) = 2.22; (ii) any complex containing Pol32 also has an extremely asymmetric shape; (iii) the results of these studies are independent of concentration (varied between 0.1-20 microm); (iv) all complexes are monomeric under the conditions studied (up to 20 microm). Moreover, a two-hybrid analysis of the Pol32 subunit did not detect a Pol32-Pol32 interaction in vivo. Therefore, we conclude that the assembly structure of Pol delta is that of a monomer.     

Isolation and characterization of a novel postsynaptic/cytotoxic neurotoxin from Daboia russelli russelli venom.

A postsynaptic neurotoxin was purified from Daboia russelli russelli venom using gel filtration, ion-exchange chromatography and reverse-phase high-performance liquid chromatography. The N-terminal sequence, molecular mass and pharmacological activities of the neurotoxin/cytotoxin indicate that it is a short-chain neurotoxin like that found in Elapid venom. This is the first report on the presence of such a postsynaptic neurotoxin from D. r. russelli venom.     

Shear-induced alignment of self-associated hemoglobin in human erythrocytes: small angle neutron scattering studies

Small angle neutron scattering (SANS) was performed on suspensions of actively metabolising human erythrocytes in the constant shear field induced by a Couette cell. The SANS pattern recorded on a two-dimensional detector was a function of the shear rate; at zero shear, the SANS pattern had radial symmetry around the direction of the beam. The radial average of the SANS pattern consisted of a broad intensity maximum superimposed on a decay. The intensity maximum at q = 0.1 Å^-1 was attributed to isotropically oriented self-associated complexes of the tetrameric oxygen transport protein hemoglobin inside the erythrocytes. A flow curve of the cell suspension was used to identify at what shear rate a suspension of uniaxially oriented ellipsoidal cells is produced. The radial symmetry of the SANS patterns persisted until the shear rate was sufficient to produce a suspension of uniaxially oriented ellipsoidal cells. Again, an intensity maximum was present in directions parallel and orthogonal to the shear axis, but this intensity maximum was superimposed upon quite different intensity decays in each direction from that of the primary neutron beam. The angular range of the SANS instrument was limited, however the results from shear-induced structural changes is consistent with a model that involves hemoglobin complexes that are aligned with respect to the plasma membranes of the elongated cells.