Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain

Hiroshi Tokumitsu, Akihiro Mizutani, Seiji Nomura, Masato Watanabe, Hiroyoshi Hidaka

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6 Citations (Scopus)


Heat stable calmodulin-binding protein has been purified from Triton X-100 soluble particulate fraction of bovine brain. Considerable purification was achieved with calmodulin coupled Sepharose 4B affinity chromatography. SDS-PAGE of the purified protein revealed the apparent homogeneity being 92 % at Mr 81,000. Isoelectric focusing of purified 81K protein gave isoelectric point of 4.3. The amino acid composition was notable for high contents of acidic amino acids (15.0 mol% of glutamic acid and 8.1 mol% of aspartic acid) and 17.4 mol% of alanine. On alkaline 1 M urea gel electrophoresis, mobility of the purified 81K protein in the presence of Ca2+ and calmodulin became lower than 81K protein alone toward the anode; however, Ca2+ solely did not affect the mobility of this protein. Similarly, S-100 protein and troponin C showed the interaction with 81K protein and a decrease of mobility in the presence of Ca2+ in alkaline urea PAGE. Binding assay of 125I-labeled calmodulin revealed that 81K protein could bind to an equimolar of 125I-calmodulin as apparent dissociation constant (Kd) of 0.65 × 10-6M.

Original languageEnglish
Pages (from-to)581-588
Number of pages8
JournalBiochemical and Biophysical Research Communications
Issue number1
Publication statusPublished - Aug 30 1989
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology


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