Chemical Characterization of the Stmcture of Cholera Toxin and Its Natural Toxoid

Primary structure of the B subunit of cholera enterotoxin

“…All of the sequence data that we have obtained, both from long sequenator runs and from enzymatic digestion, are internally consistent and are in agreement with the sequence of residues l-42 as determined by Kurosky et al [23] and with the sequences around the half-cystine residues which were determined by Lai et al [24].…”

Isolation and characterization of a precursor form of the ‘A’ subunit of cholera toxin

“…The a-chain sequence obtained was essentially identical to that in [ 161 except that we have identified position 2 to be Asn rather than Asp. The y-chain amino-terminal sequence is uniquely different from that of the o-chain [6,16]. Moreover, carboxyl-terminal analysis of the M, 29 500 component gave results which were identical to the ychain and identified leutine as carboxyl-terminus.…”

“…Strikingly, under reducing conditions, a mass species ofMr 29 500 that wasM, 5500 larger than the crchain was identified in pool B2 of the G-75 column ( fig.2B). Identical treatment of standard toxin preparations did not demonstrate the occurrence of the M, 29 500 species (not shown; see [6]) The first peak (A or Bl) that eluted from the G-75 column was a contaminant that contained40&60%carbohydrate (by weight), as judged by reaction with anthrone, and 30% protein (uncorrected for water) as determined by amino acid analysis. When tested by Ouchterlony double diffusion the contaminant formed a faint precipitin line with cholera anti-toxin and appeared antigenically identical to cholera toxin.…”

“…Residues obtained from the 890B Beckman sequencer were identified by high performance liquid chromatography (HPLC) employing Cl 8 reversed-phase chromatography (Beckman/Altex) [ 121, by gas chromatography, and by amino acid analysis after back-hydrolysis of the phenylthiohydantoin (Pth) amino acids [ 131. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis was essentially according to [6]. The preparation of anti-subunit A and anti-toxin sera and the method of Ouchterlony double diffusion were as in [ 141.…”

“…The A subunit is composed of 2 chains, a! (M, 24 000) and y (M, 5500) that are held together by a single disulfide [2,5,6].…”