Copurification of two holoenzyme-forming Calcium/Calmodulin-dependent protein kinase II isoforms as holoenzyme from porcine stomach.
Gastric acid secretion is conveyed by different signal transduction pathways, among these being the muscarinic receptor M(3)-mediated acid secretion. There is some evidence that CaMkinase II is involved in the acetylcholine-conveyed acid release. The apparent CaMkinase II-isoenzymes gamma and delta were purified as a holoenzyme from homogenate of pig gastric mucosa to apparent homogeneity. The chromatographical steps comprised cationic exchanger chromatography, calmodulin affinity chromatography, anionic exchanger chromatography, and gel filtration. The CaMkinase II showed an apparent molecular mass of 332 +/- 17.3 kDa composed of 59- and 61-kDa subunits. The latter was characterized by a polyclonal antibody directed against CaMkinase II-delta. The purified CaMkinase II showed autophosphorylation and Ca(2+)/calmodulin-dependent activation (K(0. 5) = 5 nM). Moreover, the enzyme showed inhibition by the potent CaMkinase II inhibitor KN-62 in a dose-dependent manner. Addition of purified CaMkinase II inhibits the endogenous phosphorylation of a 105-kDa protein in the NaCl/Nonidet P-40 soluble fraction of the microsomal fraction of pig gastric mucosa. Our results suggest that CaMkinase II may regulate other protein kinases or phosphoprotein phosphatases, possibly by controlling acid production.
Fährmann
M
M
Muscle Physiology Group, University of Münster, Hindenburgplatz 55, Münster, D-48143, Germany. fahrman@uni-muenster.de
Pfeiffer
A
A
D000818:Animals; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005753:Gastric Mucosa / Q000201:enzymology; D020035:Holoenzymes / Q000302:isolation & purification; D015151:Immunoblotting; D007527:Isoenzymes; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology; D010766:Phosphorylation; D012685:Sepharose / Q000378:metabolism; D012965:Sodium Chloride / Q000378:metabolism; D013552:Swine
D020035:Holoenzymes; D007527:Isoenzymes; D012965:Sodium Chloride; D012685:Sepharose; C049225:Superose 12; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases