Anisodamine causes the changes of structure and function in the transmembrane domain of the Ca(2+)-ATPase from sarcoplasmic reticulum.
The effects of anisodamine on the Ca(2+)-ATPsae of sarcoplasmic reticulum (SR) were investigated by using differential scanning calorimetry to measure the ability of anisodamine to denature the transmembrane domain and the cytoplasmic domain. Anisodamine significantly altered the thermotropic phase behaviors of the transmembrane domain of purified Ca(2+)-ATPase. Specifically, the melting temperature of the transmembrane domain moved toward lower temperatures with the concentrations of anisodamine increasing and the thermotropic phase peak was abolished at 10 mM, indicating that the stabilized structure of the transmembrane domain in the presence of Ca2+ could be destabilized by anisodamine. Decreases of the intrinsic fluorescence and increases of the extrinsic fluorescence of ANS, a fluorescent probe, showed the exposure of tryptophan and hydrophobic region, respectively, suggesting again that anisodamine caused a less compact conformation in the transmembrane domain. A marked inhibition of the Ca2+ uptake activity of SR Ca(2+)-ATPase was observed when the addition of anisodamine. The drug did not affect the cytoplasmic domain of the enzyme and only slightly decreased the ATPase activity of the enzyme at concentrations up to 10 mM. This was likely due to the destabilized protein transmembrane domain. To sum up, our results revealed that anisodamine interacted specifically with the transmembrane domain of SR Ca(2+)-ATPase and inhibited the Ca2+ uptake activity of the enzyme.
Pang
Yu-Hong
YH
National Laboratory of Biomacromolecules, Institute of Biophysics, Academia Sinica, Beijing 100101, China.
Chen
Jian-Wen
JW
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002462:Cell Membrane / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012991:Solanaceous Alkaloids / Q000494:pharmacology*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D012991:Solanaceous Alkaloids; C003922:anisodamine; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D000252:Calcium-Transporting ATPases