Jona I, Martonosi A
Biochem J. 1986 Mar 1;234(2):363-71. doi: 10.1042/bj2340363.
The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.
在天然的和异硫氰酸荧光素（FITC）标记的肌浆网囊泡中，分析了Ca2+、镧系离子（Gd3+、La3+和Pr3+）以及膜电位对色氨酸和共价结合的荧光素荧光的影响。Ca2+和镧系离子与Ca2+-ATP酶的结合增加了色氨酸的荧光强度，降低了FITC的荧光强度；这些效应与阳离子浓度的相关性与Ca2+-ATP酶的高亲和力Ca2+结合位点参与阳离子诱导的荧光变化一致。FITC标记的肌浆网囊泡的荧光也受离子取代诱导的膜电位变化的影响。膜内正电位增加，而膜内负电位降低结合的FITC的荧光。还观察到色氨酸荧光较小的电位依赖性变化。根据Ca2+-ATP酶的两种主要构象（E1和E2）讨论了Ca2+、镧系离子和膜电位对色氨酸和FITC荧光的影响，这两种构象在Ca2+转运过程中被认为会交替出现。这些观察结果支持了[Dux、Taylor、Ting-Beall和Martonosi（1985年）《生物化学杂志》260，11730 - 11743]的观点，即钒酸盐诱导的Ca2+-ATP酶晶体代表E2构象，而Ca2+和镧系离子诱导的晶体代表该酶的E1构象。
