Development of a novel photoreactive calmodulin derivative: cross-linking of purified adenylate cyclase from bovine brain.
A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. 125I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca2+-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca2+-dependent stimulation of adenylate cyclase activity. 125I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca2+ dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with 125I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.
Department of Pharmacology, University of Michigan, Ann Arbor 48109.
J K
JK
Harrison
R G
RG
Lawton
M E
ME
Gnegy
D000262:Adenylyl Cyclases / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin; D002417:Cattle; D003432:Cross-Linking Reagents; D008970:Molecular Weight; D009596:Nitrophenols; D010777:Photochemistry; D011773:Pyruvates
D002147:Calmodulin; D003432:Cross-Linking Reagents; D009596:Nitrophenols; D011773:Pyruvates; C061076:4-nitrophenyl 3-diazopyruvate; D000262:Adenylyl Cyclases