利用共振能量转移对肌浆网（Ca(2+)-Mg(2+)-ATP酶）上的半胱氨酸-344进行定位

Localization of Cys-344 on the (Ca(2+)-Mg(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.

作者信息

Mata A M, Stefanova H I, Gore M G, Khan Y M, East J M, Lee A G

机构信息

SERC Centre for Molecular Recognition, University of Southampton, UK.

出版信息

Biochim Biophys Acta. 1993 Apr 8;1147(1):6-12. doi: 10.1016/0005-2736(93)90309-n.

DOI:10.1016/0005-2736(93)90309-n

PMID:8466933

Abstract

4-Bromomethyl-6,7-dimethoxy-coumarin labels the (Ca(2+)-Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum at Cys-344. Resonance energy transfer has been used to measure the distance between this site and Lys-515 labelled with fluorescein isothiocyanate as about 37 A. The height of Cys-344 above the phospholipid/water interface has been measured by resonance energy transfer for the ATPase reconstituted into bilayers containing fluorescein-labelled phosphatidylethanolamine; the height was found to be about 45 A. None of these distances was found to alter on changing pH, or on addition of Mg2+, Ca2+ or vanadate. Quenching of the fluorescence of the coumarin-labelled ATPase with KI suggested that the fluorophore is not fully exposed on the ATPase.

摘要

4-溴甲基-6,7-二甲氧基香豆素标记骨骼肌肌浆网（Ca(2+)-Mg(2+)-ATP酶）的半胱氨酸-344位点。共振能量转移已用于测量该位点与用异硫氰酸荧光素标记的赖氨酸-515之间的距离，约为37埃。通过共振能量转移测量了重构到含有荧光素标记的磷脂酰乙醇胺的双层膜中的ATP酶上半胱氨酸-344在磷脂/水界面上方的高度；发现该高度约为45埃。在改变pH值、添加Mg2+、Ca2+或钒酸盐时，未发现这些距离有任何变化。用碘化钾淬灭香豆素标记的ATP酶的荧光表明，荧光团在ATP酶上没有完全暴露。