ADP-ribosylation by cholera toxin of membranes derived from brain modifies the interaction of adenylate cyclase with guanine nucleotides and NaF.
We have developed a method to ADP-ribosylate the stimulatory guanine nucleotide-binding protein of adenylate cyclase (GS) in brain membranes by using cholera toxin. In particular, we used isonicotinic acid hydrazide and 3-acetylpyridine adenine dinucleotide to inhibit the potent NAD-glycohydrolase activity of brain membranes, and we used the detergent Triton X-100 (at 0.1%) to improve the accessibility of the toxin and guanine nucleotides used for supporting the ADP-ribosylation. This method reveals that GS is a very abundant protein in membranes derived from calf brain (approximately 30 pmol/mg of protein). In brain, GS exists in large excess over the previously reported amount of the adenylate cyclase catalytic subunit. The modification of GS with an ADP-ribosyl residue (a) elicits a four- to fivefold activation of adenylate cyclase by GTP, (b) increases the stabilization of adenylate cyclase by GTP, and (c) reduces adenylate cyclase activation by fluoride but does not change basal activity, activation by guanosine 5'-(beta, gamma-imido)triphosphate, or the sensitivity of adenylate cyclase to heat-induced denaturation. A correlation between ADP-ribosylation and the alterations in the activation of adenylate cyclase by guanine nucleotides and by fluoride is presented.
Tamir
A
A
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts.
Gill
D M
DM
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002421:Caudate Nucleus / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000378:metabolism*; D003067:Coenzymes; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism / Q000494:pharmacology; D007538:Isoniazid / Q000494:pharmacology; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives / Q000494:pharmacology; D009244:NAD+ Nucleosidase / Q000037:antagonists & inhibitors; D012969:Sodium Fluoride / Q000494:pharmacology*
D003067:Coenzymes; D006150:Guanine Nucleotides; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; C005941:3-acetylpyridine adenine dinucleotide; D012969:Sodium Fluoride; D002772:Cholera Toxin; D009244:NAD+ Nucleosidase; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D007538:Isoniazid