Identification of a calmodulin-binding protein that co-purifies with the regulatory subunit of brain protein kinase II

D. Sarkar, J. Erlichman, C. S. Rubin

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88 Scopus citations

Abstract

Highly purified preparations of cytosolic brain RII contain a tightly associated polypeptide with a M(r) of 75,000 (P75). When purified brain and heart RII were preincubated with Ca2+ and 125I-calmodulin and then were subjected to polyacrylamide gel gelectrophoresis under nondenaturing conditions a major calmodulin-binding component was found only in the brain RII sample. The calmodulin-binding activity exhibited a higher sedimentation coefficient (9 S) than free RII (5 S) indicating that it might be a complex of P75 and RII dimers. This possibility was investigated using two specific monoclonal antibodies. Western blot analyses revealed that monoclonal antibody 918 exclusively bound to P75 in the brain RII preparation while monoclonal antibody 107 complexed RII. The calmodulin-binding component was noncovalently labeled with 125I-calmodulin separated from excess free RII by polyacrylamide gel electrophoresis under nondenaturing conditions. The identity of the polypeptides comprising the binding protein was subsequently established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Polypeptides with M(r) values of 55,000 and 75,000 that bound monoclonal antibodies 107 and 918, respectively, were observed. Thus the calmodulin-binding component in brain RII preparations is a complex containing an RII dimer and one or two molecules of P75. P75 was also present in high concentrations in Triton X-100 extracts prepared from cerebral cortex and liver membranes. P75 is phosphorylated by both cAMP-dependent and calcium-phospholipid-activated protein kinases.

Original languageEnglish (US)
Pages (from-to)9840-9846
Number of pages7
JournalJournal of Biological Chemistry
Volume259
Issue number15
StatePublished - Jan 1 1984

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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