The conformational states of Ca2+-ATPase in sarcoplasmic reticulum (SR) vesicles with or without a thousand-fold transmembrane Ca2+ gradient have been studied by fluorescence spectroscopy and fluorescence quenching. In consequence of the establishment of the transmembrane Ca2+ gradient, the steady-state fluorescence results revealed a reproducible 8% decrease in the intrinsic fluorescence while time-resolved fluorescence measurements showed that 13 tryptophan residues in SR · Ca2+-ATPase could be divided into three groups. The fluorescence lifetime of one of these groups increased from 5.5 ns to 5.95 ns in the presence of a Ca2+ gradient. Using KI and hypocrellin B (a photosensitive pigment obtained from a parasitic fungus, growing in Yunnan, China), the fluorescence quenching further indicated that the dynamic change of this tryptophan group, located at the protein-lipid interface, is a characteristic of transmembrane Ca2+ gradient-mediated conformational changes in SR · Ca2+-ATPase.

Abbreviations: SR, sarcoplasmic reticulum; HB, hypocrellin B; Trp, tryptophan; DMSO, dimethysulfoxide; Hepes, N-2-hydroxyethyl piperazine-N′-ethanesulfonic acad; SR(5000:5), SR vesicles with 1000-fold transmembrane Ca2+ gradient; SR(50:50), SR vesicles without Ca2+ gradient; Ksv(app), apparent Stern-Volmer constant; Ksvi, Stern-Volmer constant of component i for dynamic quenching

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