Independent flexible motion of submolecular domains of the Ca2+,Mg2+-ATPase of sarcoplasmic reticulum measured by time-resolved fluorescence depolarization of site-specifically attached probes.
The Ca2+-transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum was site-specifically 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 phi = 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 phi = 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 (phi = 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+.
Suzuki
S
S
Department of Pure and Applied Sciences, University of Tokyo, Japan.
Kawato
S
S
Kouyama
T
T
Kinosita
K
K
Ikegami
A
A
Kawakita
M
M
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D001692:Biological Transport; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium / Q000493:pharmacokinetics*; D000252:Calcium-Transporting ATPases; D005454:Fluorescence Polarization; D008274:Magnesium / Q000493:pharmacokinetics; D008301:Maleimides; D011487:Protein Conformation / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D008301:Maleimides; C010854:N-(1-anilinonaphthalyl)-4-maleimide; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium