title: The binding of phosphorylase kinase to immobilized calmodulin.
abstract: The binding of phosphorylase kinase to calmodulin-Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin-Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5-5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin-Sepharose, for purification procedures at low Ca2+ concentrations (5-10 microM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100- and 200-fold, respectively, in two steps.
authors.affiliation: Institute für Physiologische Chemie, Universität-GHS-Essen, Germany.
authors.forename: H P
authors.initials: HP
authors.lastname: Jennissen
authors.forename: G
authors.initials: G
authors.lastname: Botzet
mesh_terms: D000327:Adsorption; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002846:Chromatography, Affinity; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D009132:Muscles / Q000378:metabolism; D010764:Phosphorylase Kinase / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D011817:Rabbits; D011893:Rana esculenta; D012685:Sepharose
chemical_list: D002147:Calmodulin; D008024:Ligands; D012685:Sepharose; D010764:Phosphorylase Kinase; D002118:Calcium