Binding of 9-anthroylcholine monitors the interactions of adenosine cyclic3 ',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' associa tions with substrates and effectors. Additionally, the binding of 9AC is hi ghly 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 phospho rylase kinase do not associate with 9AC, The 1:1 Fluorescent complex of the isolated catalytic subunit of cAMP-dependent protein kinase with 9AC exhib its a dissociation constant of 21 mu M. The association of the catalytic su bunit with either of the regulatory subunits, R-I and R-II, results in decr eases in the observed 9AC fluorescence that are reversed upon the addition of cAMP. The effects of MgATP and of polypeptide substrates (Kemptide, trop onin I, protamine) on the 9AC-catalytic subunit complex are consistent with a general noncompetitive model in which the interactions of 9AC and the ot her ligands with the enzyme are mutually antagonistic but not purely compet itive.