Purification and characterization of a Ca2+/calmodulin-dependent protein kinase II from hog gastric mucosa using a protein-protein affinity chromatographic technique.
A peripheral member of the Ca2+/calmodulin-dependent protein kinase II (CaMkinase II) group has been purified from hog gastric mucosa with the use of a novel affinity-chromatographic step. For the well known neural isotypes of CaMkinase II, it is proposed that the subunits form holoenzymes through a specific domain at the C-terminus called the 'association domain'. We immobilized a bacterially expressed association domain from CaMkinase II-delta-2 and used it as an affinity column. This matrix was used as the last step in a sequential enzyme purification procedure from hog gastric mucosa and yielded a homogeneous CaMkinase II which showed the typical physical and enzymatic properties of CaMkinase II. The enzyme activity showed a dependence on Ca2+ and calmodulin (apparent K0.5 = 2.7 microM and K0.5 = 0.02 microM, respectively). We found a subunit molecular mass of 61 kDa. An apparent native molecular mass of 310 kDa was calculated. The Stokes radius and the sedimentation coefficient were 6.7 nm and 11.2 S, respectively. Moreover, the isolated CaMkinase II was very well inhibited by the CaMkinase-II-specific inhibitors KN-62 (Ki = 0.52 nM) and KT5926 (Ki = 0.79 nM). The phosphorylation of several substrates revealed its multifunctionality. The purified CaMkinase II had an apparent Km for Mg-ATP of 24.0 microM and for autocamtide-II of 0.62 microM. CaMkinase II is considered as a strong candidate for regulating vesicle released quanta such as acid, neurotransmitters or insulin.
Fährmann
M
M
Medizinische Klinik und Poliklinik Bergmannsheil, Universitätsklinik der Ruhr-Universität Bochum, Germany. fahrman@uni-muenster.de
Möhlig
M
M
Schatz
H
H
Pfeiffer
A
A
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism*; D002147:Calmodulin / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005753:Gastric Mucosa / Q000201:enzymology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D013379:Substrate Specificity; D013552:Swine
D002147:Calmodulin; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008345:Manganese; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D008274:Magnesium; D002118:Calcium