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 pathway s, among these being the muscarinic receptor M-3-mediated acid secretion. T here is some evidence that CaMkinase II is involved in the acetylcholine-co nveyed acid release, The apparent CaMkinase II-isoenzymes gamma and delta w ere purified as a holoenzyme from homogenate of pig gastric mucosa to appar ent homogeneity, The chromatographical steps comprised cationic exchanger c hromatography, calmodulin affinity chromatography, anionic exchanger chroma tography, 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 w as characterized by a polyclonal antibody directed against CaMkinase II-del ta, The purified CaMkinase II showed autophosphorylation and Ca2+/calmoduli n-dependent activation (K-0.5 = 5 nM). Moreover, the enzyme showed inhibiti on by the potent CaMkinase II inhibitor KN-62 in a dose-dependent manner. A ddition of purified CaMkinase II inhibits the endogenous phosphorylation of a 105-kDa protein in the NaCl/Nonidet P-40 soluble fraction of the microso mal fraction of pig gastric mucosa, Our results suggest that CaMkinase II m ay regulate other protein kinases or phosphoprotein phosphatases, possibly by controlling acid production. (C) 2000 Academic Press.