Molecular construction of Clostridium botulinum type A toxins

Abstract

Two Clostridium botulinum type A toxic fractions, named large (L) and medium (M) toxins, were eluted from Sephadex G-200. Sucrose density gradient centrifugation resolved L toxin (2.5 x 10W to 3.0 x 108 mean lethal doses per mg of N) into two fractions, 19S and 16S. The same procedure performed at pH 8 resolved it into three fractions; the heavier two were both nontoxic and hemagglutinin positive, and the lightest one (7S) was toxic. M toxin (12S) (4.5 x 10W to 5.0 x 108 mean lethal doses per mg of N) was homogeneous in electrophoresis and centrifugation at pH 6. The latter procedure performed at pH 8 demonstrated that it dissociated into uniform 7S components. The nontoxic component of M toxin was free from hemagglutinin. M toxin alone was demonstrated in culture by sucrose density gradient centrifugation at pH 6. Dialysis of the culture supernatant resulted in partial formation of 16S toxin. Centrifugation of the crystalline toxin in 1 M NaCl demonstrated 16S toxin only. The toxic components of L, M, and crystalline toxins were antigenically identical. The nontoxic components of the crystalline and L toxins, consisting of two distinct antigens, were antigenically identical; that of M toxin was identical with one of these two antigens.

Molecular characterization of two forms of nontoxic‐nonhemagglutinin components of Clostridium botulinum type A progenitor toxins

“…of progenitor toxins HA-positive and HA-negative progenitor toxins were purified as described in section 2. As reported previously [2], the HA-positive progenitor toxin preparation is a mixture of 16S and 19S toxins and HA-negative progenitor toxin preparation is 12S toxin. These preparations were subjected to SDS-PAGE with a reducing agent ( Fig.…”

“…The supernatant was precipitated with 60% saturated ammonium sulfate. After removing RNA in the precipitate by protamine treatment [2], this preparation was subjected to a SP-Toyopearl 650M (Tosoh, Tokyo, Japan) column equilibrated with 50 mM sodium acetate buffer (pH 4.2). The column was washed with 100 ml of this solution, and the concentration of NaC1 was then increased linearly to 0.5 M. The fractions which showed both toxic and hemagglutinating activity, and the fraction which showed only toxic activity were collected separately, concentrated by 70% saturation of ammonium sulfate, and then subjected to a Sephacryl S-300 (Pharmacia Biotechnology AB, Uppsala, Sweden) column equilibrated with 50 mM sodium acetate buffer (pH 4.2) containing 0.5 M NaC1, to obtain purified HA-positive (16S and 19S) and HA-negative (12S) progenitor toxins.…”

Retrograde transport of radiolabelled botulinum neurotoxin type A to the CNS after intradetrusor injection in rats

“…Botulinum neurotoxin Α is produced by Clostridium botulinum as a protein complex composed of the 150 kDa core neurotoxin, various haemagglutinins and a protein known as the non-toxin non-haemagglutinin [39]. Three different complex species with molecular sizes of 900, 500 and 300 kDa, respectively, may be isolated from C. botulinum type A cultures [40]; however, in each case, the effect on their target cells is exerted by the free 150 kDa neurotoxin which is released after dissociation of these complexes [41]. Consequently, for the purposes of the present study, we used pure BoNT/A of 150 kDa instead of the commercial botulinum toxin formulation.…”

Botulogenic Properties of Vegetables With Special Reference to the Molecular Size of the Toxin in Them

“…Type B strains produce both 12 and 16s toxins in culture (Kozaki et al 1974). Schantz and Spero (1967) reported that type A strains produce 1 9 s toxin only in culture, whereas Sugii and Sakaguchi (1975) demonstrated that they produce only 12s toxin in culture. Lamanna and Sakaguchi (1971) proposed the term "progenitor toxin" to designate the toxin appearing first in culture and in food.…”