Binding of NAD+ by cholera toxin.
1. The Km for NAD+ of cholera toxin working as an NAD+ glycohydrolase is 4 mM, and this is increased to about 50 mM in the presence of low-Mr ADP-ribose acceptors. Only molecules having both the adenine and nicotinamide moieties of NAD+ with minor alterations in the nicotinamide ring can be competitive inhibitors of this reaction. 2. This high Km for NAD+ is also reflected in the dissociation constant, Kd, which was determined by a variety of methods. 3. Results from equilibrium dialysis were subject to high error, but showed one binding site and a Kd of about 3 mM. 4. The A1 peptide of the toxin is digested by trypsin, and this digestion is completely prevented by concentrations of NAD+ above 50 mM. Measurement (by densitometric scanning of polyacrylamide-gel electrophoretograms) of the rate of tryptic digestion at different concentrations of NAD+ allowed a more accurate determination of Kd = 4.0 +/- 0.4 mM. Some analogues of NAD+ that are competitive inhibitors of the glycohydrolase reaction also prevented digestion.
Galloway
T S
TS
Department of Biochemistry, University of Edinburgh, U.K.
van Heyningen
S
S
D000244:Adenosine Diphosphate / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive / Q000187:drug effects; D002772:Cholera Toxin / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology
D046911:Macromolecular Substances; D009243:NAD; D000244:Adenosine Diphosphate; D002772:Cholera Toxin; D009244:NAD+ Nucleosidase; D014357:Trypsin