Cholera toxin, obtained as crude culture filtrates of the bacterium Vibrio cholerae, was purified by a combination of ammonium sulphate fractionation, ion-exchange chromatography on DEAE-cellulose, and gel filtration cn Ultrogel AcA-44. The purified toxin migrated as a homogeneous protein on SD5 and native polyacrylamide gels, and activated / adenylate cyclase in rat liver homogenates. Activation of rat liver adenylate cyclase by cholera toxin was strictly dependent upon the presence of NAD +. In the absence of exogenous NAD+, homogenates or membrane preparations were refractory to the toxin as a result of high levels of endogenous NAD+ - utilising activities. Under conditions in which competition for NAD+ by endogenous enzymes was minimised, the activity of cholera toxin as a stimulator of adenylate cyclase was markedly enhanced and activation of the enzyme occurred with a detectable toxin-specific % incorporation of [~^c] into TCA-precipitable material from ^adenine U-^cj- NAD+. The results were consistent with the hypothesis that cholera toxin acts by catalysing the ADP-ribosylation of an intracellular membranebound acceptor protein, and that this modification is responsible for the toxin-induced stimulation of adenylate cyclase activity. Results are also presented which argue against the validity of extrapolating from studies of the NADase activity of cholera toxin to the mechanism of toxin action on adenylate cyclase. Under a variety of conditions, NADase activity appeared to proceed independently of the physiologically important ADP-ribosyltransferase activity. furthermore, culture filtrates of V. cholerae were shown to contain a highly active NADase distinct from cholera toxin, and the possibilities of NADase contamination, even in apparently pure toxin preparations, are emphasised.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:290517 |
| Date | January 1980 |
| Creators | Tait, R. M. |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/18655 |
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