Immunochemical study of the toxin of C1. botulinum type E]

Toxins extracted from the cells and the culture fluid of C1. butulinum type E had a similar mol wt and antigenic composition. Trypsin activation led to the appearance of an additional antigen. Coefficient of the cell toxin activation was much greater than the coefficient of activation of the toxin contained in the culture fluid.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Activation of Clostridium botulinum type E toxin purified by two different procedures

Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.     

Clostridium botulinum type C toxin

Summary The purification and crystallization of type C botulinum toxin along with its physical characteristics are described. The shape of Clostridium botulinum type C toxin molecule is globular like a pressed ball with a 7.4 nm diameter and a 4.3 urn thickness. The molecular volume is approximately 185 nl and the molecular weight is 141 000. The toxin molecule is composed of two parts, which are separable under appropriate conditions. These parts have some differences in the electrophoretic properties, amino acid distribution, immunological, and functional characteristics. The toxin molecule can be reconstituted by association of S-S bond between the two chains. The expression of the toxicity requires that the fragments of the polypeptide chain carrying the necessary information be functionally organized for the proper development of the specific tertiary structure for active conformation.     

Sporulation and C2 toxin production by Clostridium botulinum type C strains producing no C1 toxin.

All of the 8 strains that were previously assumed to be nontoxigenic Clostridium botulinum type C were re-examined for their toxigenicity and were demonstrated by trypsinization of the culture filtrates to produce C2 toxin under improved cultural conditions. One per cent glucose added to trypticase peptone medium enhanced C2 toxin production. The larger the spore population, the higher the C2 toxicity and when spore population was smaller than a level of 10(4)/ml, no C2 toxicity was demonstrated. The C2 toxin was produced only during sporulation and not during vegetative growth.     

Thermal inactivation of type E botulinum toxin

The theoretical required cooking times for inactivation of type E Clostridium botulinum toxin (5,000 ld(50) mouse units per 0.5 ml) in haddock fillets of various sizes were calculated by graphical integration of the toxin inactivation rate and heat penetration data. The results indicated that normal cooking procedures should suffice to inactivate this amount of toxin. This conclusion was substantiated by the following additional experimental observations which revealed that the original experiments had been conducted under conservative conditions. First, maximal heat stability of the toxin was found to occur at about pH 5.5, with decreasing resistance upon increasing pH. The theoretical cooking times were based on destruction of the toxin at pH 6.7. The pH of radio-pasteurized inoculated haddock, when toxin production had occurred, was on the alkaline side, at which condition the toxin is heat-labile. Second, when spoilage was discernible in radio-pasteurized inoculated haddock, the toxin titer was low, about 50 ld(50) mouse units per 0.5 ml. Third, the toxin was adequately inactivated in toxic fillets after deep-fat frying for 3 min at 375 F (190.6 C) or after pan frying for 5 min per side at 400 F (204.4 C). Fourth, in this study, residual toxin activity was assayed by intraperitoneal injection of mice. It was shown that the oral toxic dose was 50 to 100 times greater than the intraperitoneal toxic dose.     

Molecular structure and immunochemical properties of highly purified hemagglutinin from Clostridium botulinum type A

A procedure for isolation of highly purified hemagglutinin from a toxic complex of culture filtrates of Cl. botulinum type A is described. This procedure includes precipitation with (NH4)2SO4, chromatography on Sephadex G-100, G-200 and DEAE-cellulose, specific adsorption on human erythrocytes and affinity chromatography. Using polyacrylamide gel electrophoresis, it was shown that hemagglutinin is a heteropolymeric protein consisting of a monomer (Mr 53 000) and a trimer (Mr 160 000). The monomer is made up of two subunits with Mr 13 000 and one subunit with Mr 27 000 covalently linked by SS-crosslinks. The number and nature of the SS-crosslinks and SH-groups in the protein molecule were determined and a hypothetical structural model of hemagglutinin was proposed. Using immunochemical analysis, it was shown that some (but not all) serological properties of the highly purified protein from Cl. botulinum type A and of its partially purified counterpart are similar to those of hemagglutinin from Cl. botulinum type B.     

Binding properties of Clostridium botulinum type C progenitor toxin to mucins

It has been reported that Clostridium botulinum type C 16S progenitor toxin (C16S toxin) first binds to the sialic acid on the cell surface of mucin before invading cells [A. Nishikawa, N. Uotsu, H. Arimitsu, J.C. Lee, Y. Miura, Y. Fujinaga, H. Nakada, T. Watanabe, T. Ohyama, Y. Sakano, K. Oguma, The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells, Biochem. Biophys. Res. Commun. 319 (2004) 327-333]. In this study we investigated the binding properties of the C16S toxin to glycoproteins. Although the toxin bound to membrane blotted mucin derived from the bovine submaxillary gland (BSM), which contains a lot of sialyl oligosaccharides, it did not bind to neuraminidase-treated BSM. The binding of the toxin to BSM was inhibited by N-acetylneuraminic acid, N-glycolylneuraminic acid, and sialyl oligosaccharides strongly, but was not inhibited by neutral oligosaccharides. Both sialyl alpha2-3 lactose and sialyl alpha2-6 lactose prevented binding similarly. On the other hand, the toxin also bound well to porcine gastric mucin. In this case, neutral oligosaccharides might play an important role as ligand, since galactose and lactose inhibited binding. These results suggest that the toxin is capable of recognizing a wide variety of oligosaccharide structures.     

Simplified purification method for Clostridium botulinum type E toxin.

Clostridium botulinum type E toxin was purified in three chromatography steps. Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer. When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid. When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form. The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex. At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein. These values are significantly higher than those previously reported.     

Isolation and characterization of highly purified alpha-1-antitrypsin.

Highly purified human alpha-1-antitrypsin (phenotype MM) was obtained by an original method of preparative electrophoresis. The criteria of homogeneity were assured by one arc in crossed immunoelectrophoresis and one band on polyacrylamide gel. A unique N-terminal amino acid (pyroglutamic acid) and a unique C-terminal residue (lysine) were identified. Determined by gel electrophoresis, its molecular weight was 47,000 daltons.