1: Biochemistry  1989 Sep 19;28(19):7734-40 

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.

Suzuki S, Kawato S, Kouyama T, Kinosita K Jr, Ikegami A, Kawakita M.

Department of Pure and Applied Sciences, University of Tokyo, Japan.

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+.

PMID: 2532932 [PubMed - indexed for MEDLINE]