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Title: Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum
Authors: Pang, Yuhong
Li, Xiaozhu
Qin, Sanbo
Zhang, Hongjie
Chen, Jianwen
Keywords: Anisodamine
Sarcoplasmic reticulum
Ca2+-transport activity
ANS binding fluorescence
Conformational change
Issue Date: 2006
Publisher: CSIR
Abstract: The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamus niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca2+ were found to be 53.0 μM, 85.0 μM and 50.1 μM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.
Page(s): 351-359
ISSN: 0301-1208
Source:IJBB Vol.43(6) [December 2006]

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