Selective affinity chromatography with calmodulin fragments coupled to sepharose.
Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.
Ni
W C
WC
Klee
C B
CB
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002147:Calmodulin / Q000032:analysis*; D002148:Calmodulin-Binding Proteins; D002417:Cattle; D002846:Chromatography, Affinity / Q000379:methods; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004533:Egtazic Acid / Q000494:pharmacology; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D012685:Sepharose; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D004533:Egtazic Acid; D012685:Sepharose; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D009597:4-Nitrophenylphosphatase; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D014357:Trypsin