Activation and stabilization of the catalytic unit of adenylate cyclase.
The requirements for stability and activity of the catalytic unit (C) of adenylate cyclase were investigated. After solubilization of bovine brain membranes in the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonate (Chaps), the catalytic unit was separated from the stimulatory guanine-nucleotide-binding protein (Gs) by gel filtration on Ultrogel AcA-34. The partially purified C unit was rapidly inactivated at 30 degrees C; 0.25 mM-ATP stabilized activity. Although C-unit activity was dependent on Mg2+ or Mn2+, stabilization by ATP did not require bivalent cations. Activity of the Ultrogel-AcA-34-purified C unit was increased by Ca2+ plus calmodulin and by phosphatidylcholine plus lysophosphatidylcholine; activity in the presence of both activators was significantly greater than with each alone. Calmodulin plus Ca2+ and phospholipids also stabilized C unit. The column-purified C unit was activated by forskolin; the effect of forskolin was additive to those of calmodulin plus Ca2+ and phospholipids. p[NH]ppG-stimulated adenylate cyclase activity was reconstituted by mixing samples from the gel-filtration column containing Gs with C unit. Activation by Ca2+ plus calmodulin and Gs plus p[NH]ppG was additive; Ca2+ plus calmodulin did not alter the concentration of p[NH]ppG required for half-maximal activation. Results were similar with forskolin and Gs plus p[NH]ppG; the presence of one activator did not alter the effect of the other. These studies define conditions for separation of C unit and Gs from brain adenylate cyclase and demonstrate that ATP (in the absence of bivalent cations), phospholipids, calmodulin plus Ca2+, and forskolin all interact with C unit in a manner that is independent of functional Gs.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002850:Chromatography, Gel; D005576:Colforsin; D004224:Diterpenes / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D010743:Phospholipids / Q000494:pharmacology
D004224:Diterpenes; D010743:Phospholipids; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D000262:Adenylyl Cyclases; D002118:Calcium