Liu D, Kennedy S D, Knauf P A
Department of Biophysics, University of Rochester Medical Center, New York 14642, USA.
Biophys J. 1995 Aug;69(2):399-408. doi: 10.1016/S0006-3495(95)79912-X.
It has been suggested that Lys-430 of band 3, with which eosin-5-maleimide (EM) reacts, is located in the external channel through which anions gain access to the external transport site, and that EM inhibits anion exchange by blocking this channel. To test this, we have used 35Cl nuclear magnetic resonance (NMR) to measure Cl- binding to the external transport site in control and EM-treated human red blood cells. Intact cells were used rather than ghosts, because in this case all line broadening (LB) results from binding to external sites. In an NMR spectrometer with a 9.4-T magnetic field, red blood cells at 50% concentration (v/v) in 150 mM Cl- medium at 3 degrees C caused 19.0 +/- 1.2 Hz LB. Of this, 7.9 +/- 0.7 Hz was due to Cl- binding to the high affinity band 3 transport sites, because it was prevented by an apparently competitive inhibitor of anion exchange, 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS). The LB was not due to hemoglobin released from the cells, as little LB remained in the supernatant after cells were removed by centrifugation. Saturable Cl- binding remained in EM-treated cells, although the binding was no longer DNDS-sensitive, because EM prevents binding of DNDS. The lower limit for the rate at which Cl- goes from the binding site to the external medium is 2.15 x 10(5) s-1 for control cells and 1.10 x 10(5) s-1 for EM-treated cells, far higher than the Cl- translocation rate at 3 degrees C (about 400 s-1). Thus, EM does not inhibit Cl- exchange by blocking the external access channel. EM may therefore be useful for fixing band 3 in one conformation for studies of Cl- binding to the external transport site.
有人提出，与嗜酸性-5-马来酰亚胺（EM）反应的带3蛋白的赖氨酸-430位于外部通道，阴离子通过该通道进入外部转运位点，并且EM通过阻断该通道来抑制阴离子交换。为了验证这一点，我们使用35Cl核磁共振（NMR）来测量对照和EM处理的人红细胞中Cl-与外部转运位点的结合。使用的是完整细胞而非血影，因为在这种情况下，所有的谱线展宽（LB）都源于与外部位点的结合。在具有9.4-T磁场的NMR光谱仪中，3℃下在150 mM Cl-培养基中浓度为50%（v/v） 的红细胞导致19.0±1.2 Hz的谱线展宽。其中，7.9±0.7 Hz是由于Cl-与高亲和力的带3转运位点结合，因为它被阴离子交换的一种明显竞争性抑制剂4,4'-二硝基芪-2,2'-二磺酸盐（DNDS）所阻止。谱线展宽不是由于细胞释放的血红蛋白所致，因为通过离心去除细胞后，上清液中几乎没有谱线展宽。EM处理的细胞中仍存在可饱和的Cl-结合，尽管这种结合不再对DNDS敏感，因为EM会阻止DNDS的结合。对于对照细胞，Cl-从结合位点到外部介质的速率下限为2.15×105 s-1，对于EM处理的细胞为1.10×105 s-1，远高于3℃下的Cl-转运速率（约400 s-1）。因此，EM不会通过阻断外部通道来抑制Cl-交换。因此，EM可能有助于将带3固定在一种构象中，用于研究Cl-与外部转运位点的结合。
