Binding of 9-anthroylcholine monitors the interactions of adenosine cyclic 3',5'-phosphate-dependent protein kinase with MgATP, substrates, and regulatory subunits.
The isolated catalytic subunit of cAMP-dependent protein kinase and smooth muscle myosin light chain kinase undergo interactions with the fluorescent dye 9-anthroylcholine (9AC) that are responsive to the two enzymes' associations with substrates and effectors. Additionally, the binding of 9AC is highly sensitive to subtle structural or functional differences among closely related protein kinases. Skeletal muscle myosin light chain kinase and the catalytically active chymotryptic fragment of the gamma-subunit of phosphorylase kinase do not associate with 9AC. The 1:1 fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhibits a dissociation constant of 21 microM. The association of the catalytic subunit with either of the regulatory subunits, RI and RII, results in decreases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, troponin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the other ligands with the enzyme are mutually antagonistic but not purely competitive.
Malencik
D A
DA
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, USA. malencid@ucs.orst.edu
Anderson
S R
SR
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002417:Cattle; D002794:Choline / Q000031:analogs & derivatives* / Q000378:metabolism; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D014357:Trypsin; D014422:Turkeys
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; C035321:9-anthroylcholine; D000255:Adenosine Triphosphate; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin; D002794:Choline