Cabrini G, Verkman A S
J Membr Biol. 1986;90(2):163-75. doi: 10.1007/BF01869934.
The equilibrium binding mechanism and kinetics of binding of diS-C3-(5) (3,3'-dipropylthiodicarbocyanine iodide) to rabbit renal brush-border membrane vesicles (BBMV) were examined using steady-state and time-resolved fluorescence, and fluorescence stopped-flow methods. In aqueous solution, diS-C3-(5) exists as a monomer at concentrations less than 5 microM with fluorescence emission peak at 670 nm (excitation 622 nm), anisotropy r = 0.102, and lifetime tau = 1.2 nsec (23 degrees C). Upon addition of increasing BBMV (voltage clamped to 0 mV using K+/valinomycin), the 670 nm emission peak decreases, corresponding to formation of a nonfluorescent membrane dimer, and subsequently a new emission peak at 695 nm increases, corresponding to membrane monomer. Dynamic depolarization studies show that aqueous diS-C3-(5) rotation is unhindered with a rotational rate R = 0.57 nsec-1 while membrane monomer is hindered with steady-state anisotropy r = 0.190, lifetime tau = 2.1 nsec, R = 0.58 nsec-1 and limiting anisotropy r infinity = 0.11. Based on equilibrium fluorescence titrations, the membrane monomer-dimer (M-D) dissociation constant, Kd = [M]2/[D][BBMV], is 0.0013, where BBMV is expressed as membrane phospholipid concentration. Three distinct kinetic processes are identified by stopped-flow experiments in which BBMV are mixed with diS-C3-(5). There is rapid binding of diS-C3-(5) to the membrane to form bound monomer with a 6-msec exponential time constant. The membrane monomer at the membrane outer surface then aggregates to form bound dimer at the outer surface with a concentration independent time constant of 30 msec. The overall dimerization reaction probably consists of a rate-limiting reorientation process (30 msec) followed by a rapid dimerization which occurs on a nanosecond time scale. Finally, there is a 0.8 to 1 sec translocation of membrane dimer between symmetric sites at the inner and outer membrane surfaces. The translocation reaction is the step which is probably sensitive to changes in transmembrane electrical potential.
采用稳态荧光、时间分辨荧光和荧光停流法研究了二硫代 - C3 - (5)（3,3'-二丙基硫代二碳菁碘化物）与兔肾刷状缘膜囊泡（BBMV）结合的平衡结合机制及动力学。在水溶液中，当浓度低于5 μM时，二硫代 - C3 - (5)以单体形式存在，荧光发射峰位于670 nm（激发波长622 nm），各向异性r = 0.102，寿命τ = 1.2 ns（23℃）。加入逐渐增加的BBMV（用K⁺/缬氨霉素将电压钳制在0 mV）后，670 nm发射峰降低，这对应于非荧光膜二聚体的形成，随后695 nm处出现新的发射峰并增强，这对应于膜单体。动态去极化研究表明，水溶液中二硫代 - C3 - (5)的旋转不受阻碍，旋转速率R = 0.57 ns⁻¹，而膜单体的旋转受到阻碍，稳态各向异性r = 0.190，寿命τ = 2.1 ns，R = 0.58 ns⁻¹，极限各向异性r∞ = 0.11。基于平衡荧光滴定，膜单体 - 二聚体（M - D）解离常数Kd = [M]²/[D][BBMV]为0.0013，其中BBMV以膜磷脂浓度表示。通过停流实验确定了三个不同的动力学过程，即BBMV与二硫代 - C3 - (5)混合时的过程。二硫代 - C3 - (5)快速与膜结合形成结合单体，指数时间常数为6 ms。膜外表面的膜单体随后聚集形成外表面的结合二聚体，浓度无关的时间常数为30 ms。整体二聚化反应可能由限速重排过程（30 ms）和随后在纳秒时间尺度上发生的快速二聚化组成。最后，膜二聚体在内膜和外膜表面的对称位点之间存在0.8至1秒的转位。转位反应可能是对跨膜电位变化敏感的步骤。
