Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine.
The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-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 Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, 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.
Pang
Yuhong
Y
Institute of Biophysics, Academy Sinica, 15 Datun Road, Chaoyang District, Beijing 100101, China.
Li
Xiaozhu
X
Qin
Sanbo
S
Zhang
Hongjie
H
Chen
Jianwen
J
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D012991:Solanaceous Alkaloids / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence
D004791:Enzyme Inhibitors; D012991:Solanaceous Alkaloids; C003922:anisodamine; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D002118:Calcium