Real‐time analysis of the calcium‐dependent interaction between calmodulin and a synthetic oligopeptide of calcineurin by a surface plasmon resonance biosensor

FEBS Letters

Takano, Emiko¹  Hatanaka, Masakazu¹  Maki, Masatoshi¹ 

[1] Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto 606-01, Japan

关键词: Calmodulin;    Calcineurin;    Calcium-dependent;    Surface plasmon resonance;    Biosensor;    EDC;    N-ethyl-N′-(dimethylaminopropyl)carbodiimide;    NHS;    N-hydroxysuccinimide;    CaM;    calmodulin;    SPR;    surface plasmon resonance;    PDEA;    2-(2-pyridinyldithio)ethaneamine hydrochloride;   

DOI  :  10.1016/0014-5793(94)00965-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The calcium-dependent interaction between calmodulin (CaM) and the synthetic oligopeptide of a predicted CaM-binding region of human calcineurin A-2 was analysed with an automated surface plasmon resonance biosensor, BIAcore. The oligopeptide was immobilized to a biosensor chip via the amino-terminal cysteine residue by a thioldisulphide exchange method. The biosensor chip was regenerated by an EGTA-containing buffer after each analysis. Kinetics experiments showed that CaM bound with a high affinity to the oligopeptide in a Ca²⁺-dependent manner. The estimated rate constants of association (k _ass) and dissociation (k _diss) were 2.3 × 1O⁵ M⁻¹·s⁻¹ and 3.9 × 10⁻³ s⁻¹, respectively. The ratio of k _diss/k _ass, 1.7 × 10⁻⁸ M, was in good agreement with the dissociation constant (K _d) of 2.4 × 10⁻⁸ M determined from the equilibrium phase.

【 授权许可】

Unknown