Effect of gangliosides and substrate analogues on the hydrolysis of nicotinamide adenine dinucleotide by choleragen.
Choleragen and its A protomer catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide. NADase activity was inhibited by gangliosides GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM2 (N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), GM3 (N-acetylneuraminyl-galactosylglucosylceramide), and GD1a (N-acetylneuraminylgalactosyl-N-acetylgalactosaminyl-E1N-acetylneuraminyl]-galactosylglucosylceramide). These gangliosides also increased the intensity of the tryptophanyl fluorescence of the isolated A protomer (lambda max = 328 nm). GM1 but not GM2, GM3, and GD1a caused a "blue shift" in the fluorescence spectrum of the B protomer. These results are consistent with other evidence that the specificity of GM1 as the choleragen receptor resides in its carbohydrate moiety. The NADase activity of choleragen was similar to that of diphtheria toxin previously described [J. Kandel, R. J. Collier & D. W. Chung (1974) J. Biol. Chem. 249, 2088-2097]. As with diphtheria toxin, analogues of NAD were inhibitory, adenine being the most effective. Significant inhibition was also noted with adenosine, AMP, ADP-ribose, nicotinamide, nicotinamide mononucleotide, and NADP. NADP was hydrolyzed only slowly by choleragen. In the NADase reaction catalyzed by diphtheria toxin, water serves as an acceptor for the ADP-ribose moiety of NAD in lieu of the natural acceptor molecule, which is elongation factor II (Kandel et al., 1974). It seems probable that the natural protein acceptor for ADP-ribose in the reaction catalyzed by choleragen is adenylate cyclase or a protein component of a cyclase complex that regulates enzymatic activity.
D000225:Adenine / Q000494:pharmacology*; D000227:Adenine Nucleotides / Q000494:pharmacology; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005732:Gangliosides / Q000494:pharmacology*; D009243:NAD / Q000378:metabolism*; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors*; D009249:NADP / Q000494:pharmacology; D009536:Niacinamide / Q000494:pharmacology; D009537:Nicotinamide Mononucleotide / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014734:Vibrio cholerae
D000227:Adenine Nucleotides; D001427:Bacterial Toxins; D005732:Gangliosides; D009243:NAD; D009537:Nicotinamide Mononucleotide; D009536:Niacinamide; D009249:NADP; D009244:NAD+ Nucleosidase; D000225:Adenine