title: Separation of isozymic forms of type II calcium/calmodulin-dependent protein kinase using cation-exchange chromatography.
abstract: Three distinct isozymes of a type II calcium/calmodulin-dependent protein kinase (CaM kinase II) from rat forebrain cytosol were separated using S-Sepharose cation-exchange resin. About 90% of the applied kinase activity was recovered in three protein peaks. Each isozymic form of the kinase was purified 200-300 fold by chromatography on S-Sepharose, calmodulin-affinity and gel filtration resins. All 3 forms of CaM kinase II had apparent molecular masses of 650-700 kDa, but contained variable proportions of 50 kDa and 58-60 kDa subunits. The molar ratios of the 50 kDa/58-60 kDa kinase subunits in each holoenzyme were determined by protein staining and [125I]calmodulin-binding studies and were approximately: 6/1, 3/1 and 1/1. The isozyme containing a 3/1 ratio of subunits corresponds to the predominant form of CaM kinase II in forebrain representing 70-80% of the total activity in cytosol, whereas the other forms each represent 5-10% of the total cytosolic activity. The substrate specificities and time courses of substrate phosphorylation for the isozymes were comparable. These studies provide a basis to examine regional, subcellular, and developmental differences in the isozymic forms of CaM kinase II which may subserve different neuronal functions.
authors.affiliation: Department of Pharmacology, SUNY/Health Science Center, Syracuse 13210.
authors.forename: M L
authors.initials: ML
authors.lastname: Vallano
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002852:Chromatography, Ion Exchange; D005260:Female; D007527:Isoenzymes / Q000302:isolation & purification*; D011494:Protein Kinases / Q000302:isolation & purification*; D051381:Rats; D011919:Rats, Inbred Strains
chemical_list: D007527:Isoenzymes; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases