The Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was sitespecifically labeled with either N-(1-anilinonaphth-4-yl)maleimide (ANM) or 5-[[(iodoacetamido)-ethyl]amino]naphthalene-1-sulfonate (IAEDANS), and the segmental motion of submolecular domains of the ATPase molecule was examined by means of time-resolved and steady-state fluorescence anisotropy measurements. The ANM-binding domain showed wobbling with a rotational relaxation time φ = 69 ns in the absence of free Ca2+ without any independent wobbling of the ANM moiety. The IAEDANS-binding domain showed a significantly slower wobbling with φ = 190 ns in the absence of Ca2+. The present results demonstrated for the first time that the ATPase molecule is composed of distinct domains whose mobilities are considerably different from each other. The binding of Ca2+ to the transport site increased the segmental motion of ANM-labeled domain, leading to a phi; value of 65 ns. Solubilization of the ANM-labeled SR membranes by deoxycholate led to a further increase in the segmental flexibility (φ = 48 ns in the absence of free Ca2+), indicating that the mobility of the ANM-binding domain was considerably restricted through interaction with the membrane. The mobility of the ANM-binding domain of solubilized ATPase was also increased to some extent upon binding of Ca2+.
|Number of pages||7|
|Publication status||Published - 1989|
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