Copurification of Two Holoenzyme-Forming Calcium/Calmodulin-Dependent Protein Kinase II Isoforms as Holoenzyme from Porcine Stomach
| Autor: | Michael Fährmann, Andreas Pfeiffer |
|---|---|
| Rok vydání: | 2000 |
| Předmět: |
Calmodulin
Swine Immunoblotting Biophysics Sodium Chloride Biology Biochemistry Chromatography Affinity Copurification Affinity chromatography Microsomes Animals Phosphorylation Protein kinase A Molecular Biology chemistry.chemical_classification Dose-Response Relationship Drug Kinase Sepharose Autophosphorylation Chromatography Ion Exchange Molecular biology Isoenzymes Kinetics Enzyme chemistry Gastric Mucosa Calcium-Calmodulin-Dependent Protein Kinases Chromatography Gel biology.protein Electrophoresis Polyacrylamide Gel Calcium-Calmodulin-Dependent Protein Kinase Type 2 Holoenzymes |
| Zdroj: | Archives of Biochemistry and Biophysics. 380:151-158 |
| ISSN: | 0003-9861 |
| DOI: | 10.1006/abbi.2000.1910 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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