Liu S Q, Ries E, Knauf P A
Department of Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
J Gen Physiol. 1996 Feb;107(2):293-306. doi: 10.1085/jgp.107.2.293.
A model in which two positively-charged titratable sites enhance the affinity for anionic substrates can explain the increase in external iodide dissociation constant (K(O)(I)) with increasing pH(O) (Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996.f Gen.Physiol. 107:271-291). If sulfate binds to the same external site as I-, this model predicts that the SO(4)= dissociation constant (K(O)(S)) should also increase. The data at pH 0 8.5 to 10 fit this prediction, and the pK for the titration is not significantly different from that (pKc) for the low-pK group that affects K(O)(1). The dissociation constant for the apparently competitive inhibitor, DNDS (4,4-dinitrostilbene-2,2'-disulfonate), also increases greatly as pH(O) increases. Particularly at high pH(O), a noncompetitive inhibition by DNDS is also evident. Increasing pH(O) from 7.2 to 11.2 increases the competitive dissociation constant by 700-fold, but the noncompetitive is only increased 20-fold. The pK values for these effects are similar to pKc for K(O)(1), as expected if DNDS binds near the external transport site, but it seems likely that additional titratable groups also affect DNDS binding. The apparent affinity for external Cl- is also affected by pH(O), in a manner similar to that observed for I-. Pretreatment with the amino-selective reagent, bis-sulfosuccinimidyl suberate (BSSS), decreases the apparent Cl- affinity at pH 8.5, but two titrations are still evident, the first (lower) of which decreases the apparent C- affinity, and the second of which surprisingly increases it. Thus, the BSSS-reactive amino groups (probably Lys-539 and Lys-851) do not seem to be involved in the titrations that affect Cl- affinity. In general, the data support the concept that a positively charged amino group (or groups), together with a guanidino group, plays an important role in the binding of substrates and inhibitors at or near the external transport site.
一种模型认为，两个带正电荷的可滴定位点增强了对阴离子底物的亲和力，这可以解释随着细胞外pH值（pH(O)）升高，细胞外碘离子解离常数（K(O)(I)）增加的现象（Liu, S. J., F.-Y. Law, and P.A. Knauf. 1996. f Gen.Physiol. 107:271 - 291）。如果硫酸根与碘离子结合在相同的细胞外位点，该模型预测硫酸根解离常数（K(O)(S)）也应增加。pH值在8.5至10之间的数据符合这一预测，且滴定的pK值与影响K(O)(1)的低pK基团的pK值（pKc）无显著差异。明显的竞争性抑制剂4,4 - 二硝基芪 - 2,2'-二磺酸盐（DNDS）的解离常数也随着pH(O)升高而大幅增加。特别是在高pH(O)时，DNDS的非竞争性抑制作用也很明显。将pH(O)从7.2提高到11.2，竞争性解离常数增加700倍，但非竞争性解离常数仅增加20倍。这些效应的pK值与K(O)(1)的pKc相似，如果DNDS结合在细胞外转运位点附近，这是预期的情况，但似乎还有其他可滴定基团也影响DNDS的结合。细胞外氯离子的表观亲和力也受pH(O)影响，其方式与碘离子类似。用氨基选择性试剂双琥珀酰亚胺基辛二酸酯（BSSS）预处理会降低pH值为8.5时的表观氯离子亲和力，但仍有两个滴定明显，第一个（较低的）会降低表观氯离子亲和力，第二个则出人意料地增加它。因此，BSSS反应性氨基基团（可能是Lys - 539和Lys - 851）似乎不参与影响氯离子亲和力的滴定过程。总体而言，数据支持这样一种概念，即一个或多个带正电荷的氨基基团与一个胍基一起，在细胞外转运位点或其附近底物和抑制剂的结合中起重要作用。
