Binding of NAD + to pertussis toxin

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consis...

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Published inBiochimica et biophysica acta Vol. 1078; no. 2; pp. 155 - 160
Main Authors Lobban, Margaret D., Irons, Laurence I., van Heyningen, Simon
Format Journal Article
LanguageEnglish
Published Amsterdam Elsevier B.V 24.06.1991
Elsevier
Subjects
Bacteriology
Biological and medical sciences
Catalysis
Circular Dichroism
Dialysis
Fluorescence
Fundamental and applied biological sciences. Psychology
Microbiology
NAD - metabolism
NAD+ Nucleosidase - metabolism
Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains
Pertussis Toxin
Protein Conformation
Spectrometry, Fluorescence
Thermodynamics
Virulence Factors, Bordetella - metabolism
NAD
Stoichiometry
Circular dichroism spectrometry
Luminescence quenching
Fluorescence
Bacteria
Dissociation constant
Molecular interaction
Scatchard plot
Bordetella pertussis
Equilibrium dialysis
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Summary:The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.
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ISSN:0167-4838
0006-3002
1879-2588
DOI:10.1016/0167-4838(91)99004-C