Sulfhydryl-alkylating reagents inactivate the NAD glycohydrolase activity of pertussis toxin.
The combination of ATP, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate), and DTT (dithiothreitol) is known to promote the expression of the NAD glycohydrolase activity of pertussis toxin, which resides in the toxin's S1 subunit. By monitoring changes in electrophoretic mobility, we have found that ATP and CHAPS act by promoting the reduction of the disulfide bond of the S1 subunit. In addition, ATP, CHAPS, and DTT allowed sulfhydryl-alkylating reagents to inactivate the NAD glycohydrolase activity. In the presence of iodo[14C]acetate, the combination of ATP, CHAPS, and DTT increased 14C incorporation into only the S1 subunit of the toxin, indicating that alkylation of this subunit was responsible for the loss of activity. If iodoacetate is used as the alkylating reagent, alkylation can be monitored by an acidic shift in the isoelectric point of the S1 peptide. Including NAD in alkylation reactions promoted the accumulation of a form of the S1 peptide with an isoelectric point intermediate between that of native S1 and that of S1 alkylated in the absence of NAD. This result suggests that NAD interacts with one of the two cysteines of the S1 subunit. In addition, we found the pH optimum for the NAD glycohydrolase activity of pertussis toxin is 8, which may reflect the participation of a cysteine in the catalytic mechanism of the toxin.
Kaslow
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HR
Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles 90033.
Lesikar
D D
DD
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002793:Cholic Acids / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D007700:Kinetics; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors*; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella / Q000037:antagonists & inhibitors*
D002793:Cholic Acids; D003902:Detergents; D010566:Virulence Factors, Bordetella; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D004229:Dithiothreitol