Purification and properties of the inhibitory guanine nucleotide regulatory unit of brain adenylate cyclase.
Hormonal inhibition of adenylate cyclase is mediated by a guanine nucleotide regulatory protein (Ni) which is different from the one which mediates hormonal stimulation. There is substantial evidence that the active component of Ni (termed alpha i can be ADP-ribosylated by a toxin from Bordetella pertussis. We have found that in bovine cerebral cortex there are three proteins of similar molecular weight (39,000-41,000) which are modified by pertussis toxin. We have purified these proteins and have resolved the 41,000-dalton protein from the 40,000/39,000-dalton doublet. All three forms of pertussis toxin substrate can be isolated in free form or together with a 36,000 beta component. We have also purified this beta component. ADP-ribosylation of the three pertussis toxin substrates is greatly enhanced by the addition of the purified beta component. This makes possible an assay of beta subunit activity based on its interaction with alpha i. The three forms of pertussis toxin substrate which we have purified differ in two functions: susceptibility to ADP-ribosylation and GTPase activity. The 41,000-dalton protein is more readily ADP-ribosylated by pertussis toxin than the smaller forms. The 39,000-dalton protein has GTPase activity with a low Km (0.3 microM) for GTP. The GTPase activity can be doubled by phospholipids. The GTPase activity of the 41,000-dalton protein is almost undetectable. It is not yet known what the relationship of the forms is to each other. The smaller forms may be derived from the larger by proteolysis or it may be intrinsically different. It remains to be shown whether one of the forms represents a different type of regulatory protein which transmits a hormonal signal to effectors other than adenylate cyclase.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D037361:Adenylate Cyclase Toxin; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008970:Molecular Weight; D037342:Pertussis Toxin; D010566:Virulence Factors, Bordetella
D037361:Adenylate Cyclase Toxin; D001427:Bacterial Toxins; D046911:Macromolecular Substances; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases