Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase
Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain...
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Published in | The Journal of biological chemistry Vol. 255; no. 12; pp. 5916 - 5923 |
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Main Authors | , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Biochemistry and Molecular Biology
25.06.1980
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Subjects | |
Online Access | Get full text |
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Summary: | Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure
involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was
purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be
activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg
of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000
from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent
phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase
was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius
to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the
complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation
of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(19)70718-2 |