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The ability to chemically couple proteins to LH(N)-fragments of clostridial neurotoxins and create novel molecules with selectivity for cells other than the natural target cell of the native neurotoxin is well established. Such molecules are able to inhibit exocytosis in the target cell and have the potential to be therapeutically beneficial where secretion from a particular cell plays a causative role in a disease or medical condition. To date, these molecules have been produced by chemical coupling of the LH(N)-fragment and the targeting ligand. This is, however, not a suitable basis for producing pharmaceutical agents as the products are ill defined, difficult to control and heterogeneous. Also, the molecules described to date have targeted neuroendocrine cells that are susceptible to native neurotoxins, and therefore the benefit of creating a molecule with a novel targeting domain has been limited. In this paper, the production of a fully recombinant fusion protein from a recombinant gene encoding both the LH(N)-domain of a clostridial neurotoxin and a specific targeting domain is described, together with the ability of such recombinant fusion proteins to inhibit secretion from non-neuronal target cells. Specifically, a novel protein consisting of the LH(N)-domains of botulinum neurotoxin type C and epidermal growth factor (EGF) that is able to inhibit secretion of mucus from epithelial cells is reported. Such a molecule has the potential to prevent mucus hypersecretion in asthma and chronic obstructive pulmonary disease.

The botulinum neurotoxin endopeptidases appear to recognise their intracellular protein substrates via two distinct sites: the cleavage site sequence and a 'recognition site' motif. In the present study phage display has been employed to generate a library of vesicle-associated membrane protein (VAMP2) variants in which the toxin recognition motif (part of the SNARE motif ELDDRADA) has been modified. VAMP (1-94) was displayed on the surface of M13 bacteriophage and this fragment was recognised and cleaved by botulinum neurotoxin type B (BoNT/B). A phage-displayed library was constructed in which six residues of the recognition domain (VAMP residues 63-68; wild-type sequence LDDRAD) were randomised, and a selection method established for identifying cleaved VAMP variants. Sequence analysis of 24 clones revealed that 5 contained two acidic residues although none corresponded to the native sequence. Cleavage was reduced compared to wild-type VAMP, and cleavage of mutants containing no acidic residues was also observed. The data are discussed in relation to the substrate recognition mechanism of BoNT/B.

Clostridium botulinum neurotoxins are potently toxic proteins of 150 kDa with specific endopeptidase activity for SNARE proteins involved in vesicle docking and release. Following treatment with trypsin, a fragment of botulinum neurotoxin serotype A that lacks the C-terminal domain responsible for neuronal cell binding, but retains full catalytic activity, can be obtained. Known as the LH(N) fragment, we report the development of a recombinant expression and purification scheme for the isolation of comparable fragments of neurotoxin serotypes B and C. Expressed as maltose-binding protein fusions, both have specific proteolytic sites present between the fusion tag and the light chain to facilitate removal of the fusion, and between the light chain endopeptidase and the H(N) translocation domains to facilitate activation of the single polypeptide. We have also used this approach to prepare a new variant of LH(N)/A with a specific activation site that avoids the need to use trypsin. All three LH(N)s are enzymatically active and are of low toxicity. The production of specifically activatable LH(N)/A, LH(N)/B, and LH(N)/C extends the opportunities for exploitation of neurotoxin fragments. The potential utility of these fragments is discussed.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Previously reported data have demonstrated that the catalytically active LH(N) endopeptidase fragment of botulinum neurotoxin type A (termed LH(N)/A) can be retargeted to a range of cell types in vitro to lead to inhibition of secretion of a range of transmitters. Here, we report the synthesis of endopeptidase conjugates with in vitro selectivity for nociceptive afferents compared to spinal neurons. Chemical conjugates prepared between Erythrina cristagalli lectin and LH(N)/A are assessed in vitro and in in vivo models of pain. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LH(N)/A, or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material is demonstrated. The duration of action of inhibition of neurotransmitter release by the conjugate in vitro is also assessed and is comparable to that observed with Clostridium botulinum neurotoxin. Selectivity of targeting and therapeutic potential have been confirmed by in vivo electrophysiology studies. Furthermore, the analgesic properties of the conjugate have been assessed in in vivo models of pain and extended duration effects observed. These data provide proof of principle for the concept of retargeted clostridial endopeptidases as novel analgesics.

C3 exoenzyme from Clostridium botulinum (C3bot1) ADP-ribosylates and thereby inactivates Rho A, B and C GTPases in mammalian cells. The structure of a tetragonal crystal form has been determined by molecular replacement and refined to 1.89 A resolution. It is very similar to the apo structures determined previously from two different monoclinic crystal forms. An objective reassessment of available apo and nucleotide-bound C3bot1 structures indicates that, contrary to a previous report, the protein possesses a rigid core formed largely of beta-strands and that the general flexure that accompanies NAD binding is concentrated in two peripheral lobes. Tetragonal crystals disintegrate in the presence of NAD, most likely because of disruption of essential crystal contacts.

The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase that ADP-ribosylates not only RhoA-C but also RhoE/Rnd3. In this study we have crystallized and determined the structure of C3stau2 in both its native form and in complex with NAD at 1.68- and 2.02-A resolutions, respectively. The topology of C3stau2 is similar to that of C3bot1 from Clostridium botulinum (with which it shares 35% amino acid sequence identity) with the addition of two extra helices after strand β1. The native structure also features a novel orientation of the catalytic ARTT loop, which approximates the conformation seen for the “NAD bound” form of C3bot1. C3stau2 orients NAD similarly to C3bot1, and on binding NAD, C3stau2 undergoes a clasping motion and a rearrangement of the phosphate-nicotinamide binding loop, enclosing the NAD in the binding site. Comparison of these structures with those of C3bot1 and related toxins reveals a degree of divergence in the interactions with the adenine moiety among the ADP-ribosylating toxins that contrasts with the more conserved interactions with the nicotinamide. Comparison with C3bot1 gives some insight into the different protein substrate specificities of these enzymes.

Using polymerase chain reaction, the coding sequence for Erythrina cristagalli lectin (ECL) has been cloned and expressed in Escherichia coli. The amplified DNA sequence of ECL is highly homologous to that previously reported for Erythrina corallodendron lectin (ECorL), confirming the absence of introns in the ECL gene. The polypeptide sequences of ECL and ECorL have been compared and five amino acids have been identified that differentiate the two proteins. Recombinant E. cristagalli lectin (recECL) was expressed in E. coli from a genomic clone encoding the mature E. cristagalli lectin gene. Constitutive expression localised recombinant protein in inclusion bodies, which were solubilised, and recECL, subsequently refolded and purified by lactose affinity chromatography. Significant advantages were observed for purification from inclusion bodies rather than from a clone optimised to express soluble protein. A large-scale purification scheme has been developed that can prepare functional recECL from inclusion bodies with a yield of 870 mg/L culture. By the range of characterisation methods employed in this study, it has been demonstrated that recECL is functionally equivalent to native ECL obtained from the E. cristagalli plant. In addition, characterisation of the binding of radiolabelled recECL to cultured dorsal root ganglia demonstrated that recECL binds to a single pool of receptors.

Nerve growth factor (NGF) receptor binding, internalisation and transportation of NGF has been identified as a potential route of delivery for other molecules. A derivative of Clostridium botulinum neurotoxin type A (LHN) that retains catalytic activity but has significantly reduced cell-binding capability has been prepared and chemically coupled to NGF. Intact clostridial neurotoxins potently inhibit neurotransmitter release at the neuromuscular junction by proteolysis of specific components of the vesicle docking/fusion complex. Here we report that the NGF-LHN/A conjugate, when applied to PC12 cells, significantly inhibited neurotransmitter release and cleaved the type A toxin substrate. This work represents the successful use of NGF as a targeting moiety for the delivery of a neurotoxin fragment.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types by a mechanism that involves cleavage of specific components of the vesicle docking/fusion complex, the SNARE complex. A derivative of the type A neurotoxin from Clostridium botulinum (termed LH N /A) that retains catalytic activity can be prepared by proteolysis. The LH N /A, however, lacks the putative native binding domain (H C ) of the neurotoxin and is thus unable to bind to neurons and effect inhibition of neurotransmitter release. Here we report the chemical conjugation of LH N /A to an alternative cell-binding ligand, wheat germ agglutinin (WGA). When applied to a variety of cell lines, including those that are ordinarily resistant to the effects of neurotoxin, WGA-LH N /A conjugate potently inhibits secretory responses in those cells. Inhibition of release is demonstrated to be ligand mediated and dose dependent and to occur via a mechanism involving endopeptidase-dependent cleavage of the natural botulinum neurotoxin type A substrate. These data confirm that the function of the H C domain of C. botulinum neurotoxin type A is limited to binding to cell surface moieties. The data also demonstrate that the endopeptidase and translocation functions of the neurotoxin are effective in a range of cell types, including those of nonneuronal origin. These observations lead to the conclusion that a clostridial endopeptidase conjugate that can be used to investigate SNARE-mediated processes in a variety of cells has been successfully generated.

A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed. The assay is amplified by the enzymic activity of the neurotoxin’s light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products. The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml −1 (0.5 mouse 50% lethal dose ml −1 ) in approximately 5 h. The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay. Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bioassay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bioassay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin of the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bioassay. It is envisaged that such assays will prove realistic alternatives to animal-based tests.

Botulinum neurotoxins type D and F are zinc-endopeptidases with a unique specificity for VAMP/synaptobrevin, an essential component of the exocytosis apparatus. VAMP contains two copies of a nine residue motif, termed V1 and V2, which are determinants of the interaction with tetanus and botulinum B and G neurotoxins. Here, we show that V1 plays a major role in VAMP recognition by botulinum neurotoxins D and F and that V2 is also involved in F binding. Site-directed mutagenesis of V1 and V2 indicates that different residues are the determinants of the VAMP interaction with the two endopeptidases. The study of the VAMP-neurotoxins interaction suggest a pairing of the V1 and V2 segments.

Tetanus and botulinum neurotoxins type B and G are zinc-endopeptidases of remarkable specificity. They recognize and cleave a synaptic vesicle-associated membrane protein (VAMP)/synaptobrevin, an essential protein component of the vesicle docking and fusion apparatus. VAMP contains two copies of a nine-residue motif, also present in SNAP-25 (synaptosomal-associated protein of 25 kDa) and syntaxin, the two other substrates of clostridial neurotoxins. This motif was suggested to be a determinant of the target specificity of neurotoxins. Antibodies raised against this motif cross-react among VAMP, SNAP-25, and syntaxin and inhibit the proteolytic activity of the neurotoxins. Moreover, the various neurotoxins cross-inhibit each other's proteolytic action. The role of the three negatively charged residues of the motif in neurotoxin recognition was probed by site-directed mutagenesis. Substitution of acidic residues in both copies of the VAMP motif indicate that the first one is involved in tetanus neurotoxin recognition, whereas the second one is implicated in binding botulinum B and G neurotoxins. These results suggest that the two copies of the motif have a tandem association in the VAMP molecule.

Clostridium botulinum type B neurotoxin is a highly specific zinc-endopeptidase which cleaves vesicle-associated membrane protein (VAMP/synaptobrevin), a critical component of the vesicle docking/fusion mechanism. In this study, substrate residues flanking the N-terminal side of the cleavage site are shown to play a key role in enzyme substrate recognition. Two aspartate residues in this region are identified as critical determinants of the neurotoxin's specificity. These findings are discussed in relation to the mechanism by which botulinum type B neurotoxin cleaves its substrate.

Western blotting of the insulin-secreting beta-cell lines HIT-15 and RINm5F with anti-SNAP-25 (synaptosomal associated protein of 25 kDa), anti-synaptobrevin, and anti-syntaxin 1 antibodies revealed the presence of proteins with the same electrophoretic mobility as found in neural tissue. Permeabilization of both of these insulinoma cell lines to botulinum neurotoxin A by electroporation resulted, after 3 days of culture, in the loss of approximately 90% of SNAP-25 immunoreactivity. A similar permeabilization of these cells with botulinum neurotoxin B resulted in the cleavage of approximately 90% of the synaptobrevin-like immunoreactivities. Botulinum neurotoxin F also cleaved approximately 90% of the synaptobrevin-like immunoreactivity in RINm5F cells. The permeabilization of both insulinoma cells to neurotoxin A resulted in a90% inhibition of potassium-stimulated, calcium-dependent insulin release. By contrast, permeabilization of the insulinoma cell lines to neurotoxin B resulted in only a approximately 60% inhibition of potassium-stimulated insulin release in HIT-15 cells, and neither neurotoxin B nor F caused inhibition in RINm5F cells. Thus HIT-15 and RINm5F cells contain the components of the putative exocytotic docking complex described in cells derived from the neural crest. In HIT-15 cells both SNAP-25 and synaptobrevin appear to be involved in calcium-dependent insulin secretion, whereas in RINm5F cells SNAP-25 but not synaptobrevin is involved.

Botulinum neurotoxin serotype C (BoNT/C) is a 150-kDa protein produced by Clostridium botulinum, which causes animal botulism. In contrast to the other botulinum neurotoxins that contain one atom of zinc, highly purified preparations of BoNT/C bind two atoms of zinc per toxin molecule. BoNT/C is a zinc-endopeptidase that cleaves syntaxin 1A at the Lys235-Ala254 and syntaxin 1B at the Lys252-Ala253 peptide bonds, only when they are inserted into a lipid bilayer. The other Lys-Ala bond present within the carboxyl-terminal region is not hydrolyzed. Syntaxin isoforms 2 and 3 are also cleaved by BoNT/C, while syntaxin 4 is resistant. These data suggest that BoNT/C recognizes a specific spatial organization of syntaxin, adopted upon membrane insertion, which brings a selected Lys-Ala peptide bond of its carboxyl-terminal region to the active site of this novel metalloproteinase.

Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of human/rat VAMP-2 [VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (Km = 3.3 x 10(-4) M) and by its purified light subunit (Km = 3.5 x 10(-4) M). The endopeptidase displayed a pH optimum of 7.0-7.5 and was inhibited by greater than 0.2 M NaCl and greater than 0.05 M sodium phosphate. Neurotoxin which had been inactivated by dialysis against EDTA could be re-activated by incubation with various divalent cations, notably Zn2+ and Cu2+. The substrate specificity of botulinum type B neurotoxin was studied using various analogues of VAMP-2 (60-94). The neurotoxin cleaved selectively to the N-terminal side of phenylalanine and tyrosine; no activity was observed with either leucine, valine or alanine in the P'1 position. The properties of the P1 amino acid were less critical; the neurotoxin cleaving the C-terminus of glutamine, asparagine and alanine. A substrate analogue with valine in the P1 position corresponding to the sequence of rat VAMP-1 was not cleaved. The rate of cleavage of a substrate analogue representing the sequence of human VAMP-1, however, was more than twofold that of the VAMP-2 peptide. The properties and substrate specificity of botulinum type B neurotoxin suggest that the toxin represents a novel class of endopeptidase which requires a specific peptide substrate conformation for the expression of proteolytic activity.

Recent data suggest that botulinum type-B neurotoxin is a protease which acts on vesicle-associated membrane protein, isoform 2 (VAMP-2). In this report, botulinum type-B neurotoxin is shown to cleave a synthetic fragment (HV62) of VAMP-2, corresponding to the bulk of the hydrophillic domain (amino acids 33–94). The neurotoxin acts at a single site between Gln76 and Phe77. Little or no proteolytic activity by botulinum type-B neurotoxin was observed with peptides containing 7, 10 or 20 amino acids spanning the site of cleavage. The proteolytic action of neurotoxin was strongly inhibited by EDTA and o-phenanthroline whereas captopril and phosphoramidon were ineffective. A series of model peptide substrates were synthesised in order to define the smallest VAMP-2 fragment to be cleaved by botulinum type-B neurotoxin. Data obtained from these substrates suggest that the neurotoxin belongs to a novel class of zinc-endoprotease; more than 12 amino acid residues are required on both the NH2-and COOH-terminal side of the cleavage site for optimal proteolytic activity. The results demonstrate that no other components of cellular vesicles are required for the specific action of the neurotoxin on VAMP-2. The data further show that the highly specific action of the neurotoxin is not dictated solely by the properties of the amino acid residues at the cleavage site but is also dependent on amino acid sequences distal to its site of action.

Clostridium botulinum type A neurotoxin (BoNTA) slowly inhibits the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. The effects of BoNTA on PC12 cells are shown to persist for several days and in subsequent cell generations. Examination of the molecular form of cell-associated 125I-labelled BoNTA after 96h incubation within PC12 cells showed virtually no detectable degradation of either the heavy or light chains of the neurotoxin. The data suggest that BoNTA is relatively stable within the PC12 cell with a half-life in the order of several days. More than 50% of the membrane-associated BoNTA within the cell exists in the form of high molecular weight, disulphide-linked aggregates and these appear to be enriched in the light chain of the neurotoxin. A possible role in action of BoNTA for these aggregates is discussed.

The botulinum neurotoxin types primarily involved in human illness are types A, B, E and F. Type A neurotoxin is the most intensively studied of the neurotoxins. However the antigenic sites remain unknown. The aim of the current project is to study the structure and determine the location of the antigenic sites of the type A neurotoxin.

(a) Clostridium botulinum type-A neurotoxin (BoNTA) inhibited the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37 degrees C, a neurotoxin concentration of approximately 0.12 +/- 0.03 microM was required to inhibit 50% of the calcium-dependent noradrenaline release. (b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose-dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I-labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high-affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non-specific binding to the cell membrane or via a low-concentration low-affinity acceptor molecule. (c) A study of the long-term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0.3 microM) for 24 h was reduced to less than 20% of control values over a subsequent 4-day period. After 8 days, release levels were significantly lower (60-65%) than control values, despite a more than 10-fold increase in the cell mass. (d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94-98%) to be associated with the cell membrane fraction. Of this, 50-80% of the BoNTA was associated with the nuclear and cell debris fraction and 11-25% was recovered in the large-granule-vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar. (e) Examination of the form of the cell-associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non-reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Publisher Summary Botulism—a frequently fatal disease affecting both humans and animals—is caused by any one of the seven antigenically different neurotoxins produced by various strains of the bacterium Clostridium botulinum. The primary site of action of all the botulinum neurotoxins is the neuromuscular junction where, following a binding step in which toxin molecules interact with acceptor sites on the presynaptic nerve surface, they enter the nerve ending and block the calcium-dependent release of neurotransmitter. Botulinum type F neurotoxin is an extremely potent neuroparalytic agent with a human lethal dose on the order of a few micrograms. Although several different bacterial strains and culture conditions are used to prepare small quantities of botulinum type F toxin, the Langeland strain of C. botulinum type F is most widely used because of the high yields of toxin obtained during growth. This chapter describes the various purification and radiolabeling procedures of C. botulinum type F neurotoxin.

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.

The entire structural gene of the Clostridium botulinum NCTC 11219 type-E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH-terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.

A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid. The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested. The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody. A sensitive immunoassay for C. botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin. The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1.

C. botulinum neurotoxins are acutely toxic materials and act by inhibiting release of the neurotransmitter acetylcholine. The specific nature of this inhibition is discussed and the preparation and purification of Type A toxin specifically for clinical use is described.

Coenzyme A linked aldehyde dehydrogenase from Escherichia coli strain B has been purified to a specific activity of 14 units/mg of protein, and initial rate and substrate analogue inhibition experiments have been performed. On the basis of these steady-state measurements, a bi-uni-uni-uni ping-pong mechanism is proposed in which NAD+ binds to the free enzyme followed by acetaldehyde. The product NADH is then released before coenzyme A (CoA) can bind, and acetyl-CoA is the final product released. A pre-steady-state time-dependent activation of the enzyme was observed when assays were initiated with NAD+. This lag phase of the reaction was studied as a function of the NAD+ concentration and found to be first order. Furthermore, the presence of NAD+ was demonstrated to be necessary to maintain the enzyme in the active conformation. Evidence that the enzyme contains two distinct NAD+ binding sites, an activator site and a catalytic site, has been obtained from pre-steady-state experiments with the NAD+ analogues AMP and 3-pyridine-carboxaldehyde adenine dinucleotide. AMP, a potent competitive inhibitor with respect to NAD+ under steady-state conditions, did not affect the rate of enzyme activation during pre-steady-state measurements. The analogue 3-pyridine-carboxaldehyde adenine dinucleotide, a potent activator of the aldehyde dehydrogenase, was a poor substrate compared with NAD+. At concentrations of this analogue that fully activated the enzyme, no alternate substrate inhibition was observed with respect to NAD+. A model incorporating two binding sites for NAD+ has been put forward to explain these observations.

1. The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the aminoterminal region of the heavy-chain disulphide-linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve-diaphragm preparations and cerebrocortical synaptosomes. 2. No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons. 3. The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di-chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia. 4. Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino-terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain. 5. A role within the neuron is implicated for a carboxy-terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.

A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl. botulinum spores inoculated into pork slurries. Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C. Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method. A total of 48 strains, consisting of 38 Cl. botulinum and 10 Cl. sporogenes (putrefactive anaerobes), and 140 slurry samples were tested. Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA. No false-positive reactions occurred with Cl. botulinum types A, C, D, E or F, or with the 10 strains of Cl. sporogenes. Toxin produced by one strain of Cl. botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA. With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.

Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity. The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit. A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit. Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit. This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin. The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined. The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action. It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site.

1. 1. Hexokinase I has been partially purified from pig brain tissues and an initial rate study performed. 2. 2. A recent study of Vowles & Easterby (1979), of hexokinase I from pig heart tissue has shown the glucose-6-P inhibited enzyme to be unaffected by inorganic orthophosphate. 3. 3. In this report we demonstrate that pig brain and pig heart type I hexokinases are distinct iosyzmes, differing markedly in their regulatory properties in that glucose-6-P inhibition of brain hexokinase is reversed by inorganic orthophosphate.

International audience; 1. The abilities of botulinum neurotoxins, types A and B (single and two‐chain forms) to inactivate an intraneuronal component required for transmitter release were quantified in a phrenic‐nerve‐diaphragm preparation, cerebrocortical synaptosomes or the buccal ganglion of Aplysia californica and compared with the mouse toxicity assay. 2. Homogeneous preparations of the individually renatured polypeptide chains of both toxin types showed low residual toxicity in the whole animal and had no effect on neurotransmission in all three systems, when tested singly. 3. Mixtures of individually renatured heavy chain, from type A or B, and either light chain proved very effective in blocking the evoked release of acetylcholine when bath‐applied to the buccal ganglion of Aplysia whilst they were relatively inactive on mammalian nerve terminals, indicating a less efficient uptake of the polypeptides in the latter. 4. When renatured together, the homologous, but not the heterologous, chains of each toxin type yielded toxic, disulphide‐linked two‐chain species. 5. A role for the heavy chain alone in acceptor recognition and membrane translocation was implicated by the blockade of acetylcholine release produced when light chain was applied to a ganglion of Aplysia previously bathed in heavy chain and washed extensively. No blockade was observed when the order of application of the two chains was reversed. 6.These findings are discussed in the context of the intracellular requirement for both the constituent toxin chains for toxicity, and in the apparent need for these chains to be linked via a disulphide bond for uptake in rodents but not in Aplysia.

The binding, internalization, and inhibition of transmitter release by botulinum neurotoxin (BoNT) was investigated using the intact toxin, its heavy (HC) or light (LC) chains, and a proteolytic fragment thereof. In Aplysia neurons, blockade of acetylcholine release upon external application of BoNT types A or E was prevented by reducing the temperature to 10 degrees C, due to arresting intoxication at the membrane binding step. At this low temperature, type A HC, H2 (comprised of the N-terminal of HC), or H2L (H2 disulfide-linked to LC) antagonized the neuroparalytic action of BoNT A or E, indicating that the latter bind saturably to common ecto-acceptor via the H2 region. In contrast, H2L was unable to counteract BoNT-induced paralysis at the murine neuromuscular junction. In accordance with this species difference, unlike native BoNT, saturable binding of 125I-labeled H2L could not be detected in mammalian peripheral or central nerve terminals. Possibly, more stringent structural requirements form the basis of the toxin's greater effectiveness in inhibiting neurotransmission at mouse nerve muscle synapses than Aplysia nerve terminals. In further identification of functional domains in the toxin, an unprocessed single-chain form of BoNT type E was found to be ineffective when applied extra- or intracellularly to Aplysia neurons. Notably, bath application of the latter to a neuron preinjected with HC, but not H2L or LC, resulted in a blockade of release. This shows that the single-chain species can become internalized and requires, not only LC, but also processed HC for its inhibitory action; consistently, the proteolyzed form of BoNT E was active.

A procedure has been developed for purification of the tripartite anthrax-toxin components. This involves sequential high-performance anion-exchange, gel-filtration and hydrophobic-interaction chromatography. From an initial culture volume of 15 litres, typical yields of 8 mg of protective antigen, 13 mg of lethal factor and 7 mg of oedema factor are produced to higher degrees of purity than have previously been achieved by conventional chromatographic techniques.