A proper transmembrane Ca2+ gradient is essential for the stimulation of adenylate cyclase activity by stimulatory GTP-binding protein.
Stimulatory GTP-binding Protein (Gs) and adenylate cyclase prepared from bovine brain cortices were co-reconstituted into asolectin vesicles with or without 1000-fold transmembrane Ca2+ gradient. The results showed that both basal activity and Gs-stimulated activity of adenylate cyclase were highest in proteoliposomes with a transmembrane Ca2+ gradient similar to physiological condition (1 microM Ca2+ outside and 1 mM Ca2+ inside) and lowest when the transmembrane Ca2+ gradient was in the inverse direction. Such a difference could be diminished following dissipation of the transmembrane Ca2+ gradient by A23187. Comparable conformational changes of Gs in proteoliposomes were also observed when Gs was labeled with the fluorescence probe, acrylodan. These results may indicate that a proper transmembrane Ca2+ gradient is essential not only for higher adenylate cyclase activity but also for its stimulation by Gs.
National Laboratory of Biomacromolecules, Chinese Academy of Sciences, Beijing.
Y
Y
Huang
G
G
Fan
F
F
Yang
D000249:Adenosine Monophosphate; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D008081:Liposomes; D008564:Membrane Potentials; D010713:Phosphatidylcholines; D010743:Phospholipids; D011487:Protein Conformation; D011510:Proteolipids; D015398:Signal Transduction; D013050:Spectrometry, Fluorescence; D013268:Stimulation, Chemical
D015220:Calcium Channels; D008081:Liposomes; D010713:Phosphatidylcholines; D010743:Phospholipids; D011510:Proteolipids; C020485:proteoliposomes; D000249:Adenosine Monophosphate; C024990:asolectin; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D002118:Calcium