TY - JOUR
T1 - Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain
AU - Tokumitsu, Hiroshi
AU - Mizutani, Akihiro
AU - Nomura, Seiji
AU - Watanabe, Masato
AU - Hidaka, Hiroyoshi
N1 - Funding Information:
We thank Dr. M. Hagiwara for his helpful discussion. was supported in part by a Grant-Aid for Scientific research Ministry of Education, Science and Culture, Japan.
PY - 1989/8/30
Y1 - 1989/8/30
N2 - 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.
AB - 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.
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U2 - 10.1016/0006-291X(89)92177-3
DO - 10.1016/0006-291X(89)92177-3
M3 - Article
C2 - 2775288
AN - SCOPUS:0024415868
VL - 163
SP - 581
EP - 588
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
SN - 0006-291X
IS - 1
ER -