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

Sei Ichi Suzuki, Suguru Kawato, Tsutomu Kouyama, Kazuhiko Kinosita, Akira Ikegami, Masao Kawakita

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

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+.

Original languageEnglish
Pages (from-to)7734-7740
Number of pages7
JournalBiochemistry
Volume28
Issue number19
Publication statusPublished - 1989
Externally publishedYes

Fingerprint

Ca(2+) Mg(2+)-ATPase
Depolarization
Sarcoplasmic Reticulum
Fluorescence
Adenosine Triphosphatases
Membranes
Molecules
Fluorescence Polarization
Deoxycholic Acid
Calcium-Transporting ATPases
maleimide
Relaxation time
Muscle
Skeletal Muscle
Anisotropy
Rabbits

ASJC Scopus subject areas

  • Biochemistry

Cite this

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, Sei Ichi; Kawato, Suguru; Kouyama, Tsutomu; Kinosita, Kazuhiko; Ikegami, Akira; Kawakita, Masao.

In: Biochemistry, Vol. 28, No. 19, 1989, p. 7734-7740.

Research output: Contribution to journalArticle

Suzuki, Sei Ichi ; Kawato, Suguru ; Kouyama, Tsutomu ; Kinosita, Kazuhiko ; Ikegami, Akira ; Kawakita, Masao. / 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. In: Biochemistry. 1989 ; Vol. 28, No. 19. pp. 7734-7740.
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