ADP-ribosylation of membrane proteins by bacterial toxins in the presence of NAD glycohydrolase.
The ADP-ribosylation of membrane G proteins is difficult to achieve in tissues that are rich in membrane-bound NAD glycohydrolase (NAD+ glycohydrolase, EC 3.2.2.5). For many animal species this problem can be surmounted by inhibiting NAD hydrolysis with a combination of the anti-tuberculous drug, isonicotinic acid hydrazide, and the NAD analog, 3-acetylpyridine adenine dinucleotide, which act synergistically. In their presence, the ADP-ribosylation of cholera and pertussis toxin substrates reach plateau levels even with only 10 microM NAD. Although 3-acetylpyridine adenine dinucleotide acts as a weak substrate for the toxins, it is simple to estimate its contribution to the ADP-ribosylation and thus to determine the total amount of ADP-ribosylation substrate present in a tissue sample. NAD glycohydrolases that are insensitive to isonicotinic acid hydrazide are also less sensitive to 3-acetylpyridine adenine dinucleotide, but may be inactivated by dithiothreitol. Isonicotinic acid hydrazide adenine dinucleotide, the product of an exchange reaction catalysed by NAD glycohydrolase, runs with NAD in most thin-layer chromatographic systems. It can be separated from NAD, and quantitated, if the chromatographic solvent contains benzaldehyde. Isonicotinic acid hydrazide itself inhibits NAD glycohydrolase. It need not first be converted into isonicotinic acid hydrazide adenine dinucleotide.
Gill
D M
DM
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA 02111.
Coburn
J
J
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002772:Cholera Toxin / Q000494:pharmacology*; D010856:Columbidae; D004357:Drug Synergism; D004910:Erythrocyte Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D007538:Isoniazid / Q000494:pharmacology*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009243:NAD / Q000031:analogs & derivatives* / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D037342:Pertussis Toxin; D013552:Swine; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D008565:Membrane Proteins; D010566:Virulence Factors, Bordetella; D009243:NAD; D000246:Adenosine Diphosphate Ribose; C005941:3-acetylpyridine adenine dinucleotide; D002772:Cholera Toxin; D037342:Pertussis Toxin; D009244:NAD+ Nucleosidase; D019204:GTP-Binding Proteins; D007538:Isoniazid