Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain.
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 x 10(-6) M.
Department of Pharmacology, Nagoya University School of Medicine, Japan.
H
H
Tokumitsu
A
A
Mizutani
S
S
Nomura
M
M
Watanabe
H
H
Hidaka
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification* / Q000502:physiology; D002417:Cattle; D002846:Chromatography, Affinity; D008566:Membranes / Q000032:analysis; D008970:Molecular Weight
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins