title: Purification and characterization of casein kinase I from broccoli.
abstract: Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.
authors.affiliation: Department of Biology, University of Pennsylvania, Philadelphia 19104-6018.
authors.forename: L J
authors.initials: LJ
authors.lastname: Klimczak
authors.forename: A R
authors.initials: AR
authors.lastname: Cashmore
mesh_terms: D047388:Casein Kinases; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D015151:Immunoblotting; D007546:Isoquinolines; D008970:Molecular Weight; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification*; D014675:Vegetables / Q000201:enzymology*
chemical_list: D004800:Enzymes, Immobilized; D007546:Isoquinolines; C058815:N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide; D011494:Protein Kinases; D047388:Casein Kinases