The S1 subunit of pertussis toxin catalyses the hydrolysis of NAD+ (NAD+ glycohydrolysis) and the NAD(+)-dependent ADP-ribosylation of guanine-nucleotide-binding proteins. Recently, the S1 subunit of pertussis toxin was shown to be photolabelled by using radiolabelled NAD+ and u.v.; the primary labelled residue was Glu-129, thereby implicating this residue in the binding of NAD+. Studies from various laboratories have shown that the N-terminal portion of the S1 subunit, which shows sequence similarity to cholera toxin and Escherichia coli heat-labile toxin, is important to the maintenance of both glycohydrolase and transferase activity. In the present study the photolabelling technique was applied to the analysis of a series of recombinant-derived S1 molecules that possessed deletions or substitutions near the N-terminus of the S1 molecule. The results revealed a positive correlation between the extent of photolabelling with NAD+ and the magnitude of specific NAD+ glycohydrolase activity exhibited by the mutants. Enzyme kinetic analyses of the N-terminal mutants also identified a mutant with substantially reduced activity, a depressed photolabelling efficiency and a markedly increased Km for NAD+. The results support a direct role for the N-terminal region of the S1 subunit in the binding of NAD+, thereby providing a rationale for the effect of mutations in this region on enzymic activity.
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June 1990
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Research Article|
June 15 1990
Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+
W Cieplak, Jr;
W Cieplak, Jr
*Laboratory of Pathobiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840, U.S.A.
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C Locht;
C Locht
†Smith Kline Biologicals, Rixensart, Belgium, Thousand Oaks, CA 91320, U.S.A.
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V L Mar;
V L Mar
‡Amgen, Inc., Thousand Oaks, CA 91320, U.S.A.
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W N Burnette;
W N Burnette
‡Amgen, Inc., Thousand Oaks, CA 91320, U.S.A.
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J M Keith
J M Keith
*Laboratory of Pathobiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rocky Mountain Laboratories, Hamilton, MT 59840, U.S.A.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1990 London: The Biochemical Society
1990
Biochem J (1990) 268 (3): 547–551.
Citation
W Cieplak, C Locht, V L Mar, W N Burnette, J M Keith; Photolabelling of mutant forms of the S1 subunit of pertussis toxin with NAD+. Biochem J 15 June 1990; 268 (3): 547–551. doi: https://doi.org/10.1042/bj2680547
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