Affinity chromatography of brain cyclic nucleotide phosphodiesterase using 3-(2-pyridyldithio)propionyl-substituted calmodulin linked to thiol-sepharose

Abstract

[3-(2-Pyridylthio)propionyl]calmodulin (PDP-CaM), an activated thiol derivative of calmodulin (CaM), was synthesized. Preparations of this derivative containing an average of 2.8 mol of substituent/mol of protein activated purified cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of native CaM. PDP-CaM was covalently coupled to free thiol-Sepharose 4B through formation of a stable mixed disulfide bond for use in affinity chromatography. The binding capacity of the disulfide-linked CaM-Sepharose for phosphodiesterase activity was proportional to substituent level up to 4 mg of CaM/mL of gel; the total capacity of the gel for binding phosphodiesterase was 4 times that of CNBr-coupled CaM-Sepharose. Quantitative recovery was achieved by desorption of both ligand and bound proteins with a reducing agent. The thiolated CaM derivative was then separated from phosphodiesterase by rapid gel filtration; the overall recovery of phosphodiesterase activity was greater than 70%. Preparations of homogeneous enzyme in good yield were obtained after a second chromatography step on CaM-Sepharose. Binding and recovery of phosphodiesterase activity were entirely reproducible, since each preparation of affinity gel was used only once. As it permits separation of interacting species in free solution, this general method may be useful with other ligands for increasing yields from affinity chromatography, particularly when dissociation of molecules in their matrix-bound conformation may be difficult to achieve.