TAILIEUCHUNG - Báo cáo khoa học: The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity

The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+ ⁄CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, , Myo1d-IQ2 and Myo1dDLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca 2+ . | ềFEBS Journal The two IQ-motifs and Ca2 calmodulin regulate the rat myosin 1d ATPase activity Danny Kohler Sandra Struchholz and Martin Bahler Institute for GeneralZoology and Genetics Westfalische Wilhelms University Munster Germany Keywords calmodulin Ca2 regulation IQ-motif myosin Myoid Correspondence M. Bahler Institut fur Allgemeine Zoologie und Genetik Westfalische Wilhelms-Universitat Schlossplatz 5 48149 Munster Germany Fax 49 251 8324723 Tel 49 251 8323874 E-mail baehler@ These authors contributed equally to this work Received 24 January 2005 revised 25 February 2005 accepted 4 March 2005 doi The light chain binding domain of rat myosin 1d consists of two IQ-motifs both of which bind the light chain calmodulin CaM . To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2 CaM binding we expressed and affinity purified the Myo1d constructs Myo1d-head Myo1d-IQ1 Myo1d-IQ2 and Myo1dDLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2 . IQ2 had a lower affinity for CaM in the absence of Ca2 than in the presence of Ca2 . The actin-activated ATPase activity of Myo1d was 75 inhibited by Ca2 -binding to CaM. This inhibition was observed irrespective of whether IQ1 IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2 -dependence we propose that Ca2 -binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif s . Interestingly a similar but Ca2 -independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2 fused directly to the Myo1d-head was rotated through 200 by the deletion of two amino acids in the lever arm a-helix N-terminal to the IQ-motif. Myosins are actin-based motors that serve a variety of cellular functions. They generally .

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