Strontium binding to sarcoplasmic reticulum Ca2+‐ATPase

FEBS Letters

Champeil, Philippe¹  Orlowski, Stéphane¹ 

[1] Unité de Recherche Associée 1290 (Centre National de la Recherche Scientifique) and Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire (Commissariat à l'Energie Atomique), Centre d'Etudes de Saclay, 91191 Gif-sur-Yvette Cedex, France

关键词: Sarcoplasmic reticulum;    Ca2+-ATPase: Strontium;    Fluorescent probe;    SR;    sarcoplasmic reticulum;    ATPase;    adenosine triphosphatase;    EGTA;    [ethylenebis(oxyethylenenitrilo)]tetraacetic acid;    EDTA;    ethylenediaminetetraacetic acid;    MOPS;    4-morpholinepropanesulfonic acid;    Tris;    tris(hydroxymethyl)aminomethane;    FITC;    fluorescein 5'-isothiocyanate;    Trp;    tryptophan;    Lys;    lysine;    NBD-Cl;    7-chloro-4-nitrobenzo-2-oxa-1;    3-diazole;    A23187;    calcimycin;    pNPP;    p-nitrophenylphosphate;    E;    EM;    EM2;    ATPase species with zero;    one or two Sr2+ ions bound to the transport sites;   

DOI  :  10.1016/0014-5793(93)80947-S

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

We investigated the consequences of Sr²⁺ binding to the transport sites of sarcoplasmic reticulum (SR) Ca²⁺-ATPase for two fluorescent conformational probes located in different regions of the ATPase. Using SR vesicles in which Lys-515 in the ATPase had been previously labeled with fluorescein 5'-isothiocyanate (FITC), we found that the Sr²⁺-induced a drop in the fluorescein fluorescence of this FITC-labeled ATPase shifted toward lower Sr²⁺ concentrations than the Sr²⁺-induced rise in Trp fluorescence for the same FITC-labeled ATPase. The curve describing the Sr²⁺-dependent rise in Trp fluorescence had a characteristic asymmetric shape, and the changes in Trp fluorescence occurred in parallel with the activation by Sr²⁺ of pNPP hydrolysis by the ATPase. Analysis of these results in terms of the simplest scheme describing the sequential binding of the two Sr²⁺ ions suggests that under the conditions of these experiments, i.e. at neutral pH in the presence of potassium, the Sr²⁺-induced rise in the Trp fluorescence mainly reflected the formation of ATPase with two ions bound to the transport sites, whereas the binding of a single Sr²⁺ ion was virtually sufficient to reduce the fluorescence of bound FITC to its minimal level.

【 授权许可】

Unknown