Jennings M L, Whitlock J, Shinde A
Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Biochem Cell Biol. 1998;76(5):807-13. doi: 10.1139/bcb-76-5-807.
Pre-steady state Cl- efflux experiments have been performed to test directly the idea that the transport inhibitor H2DIDS (4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate) binds preferentially to the outward-facing state of the transporter. Cells were equilibrated with a medium consisting of 150 mM sodium phosphate, pH 6.2, N2 atmosphere, and 80-250 microM 36Cl-. Addition of H2DIDS (10-fold molar excess compared with band 3) induces a transient efflux of Cl-, as expected if H2DIDS binds more tightly to outward-facing than to inward-facing states. The size of the H2DIDS-induced efflux depends on the Cl- concentration and is about 700,000 ions per cell at the highest concentrations tested. The size of the transient efflux is larger than would be expected if the catalytic cycle for anion exchange involved one pair of exchanging anions per band 3 dimer. These results are completely consistent with a ping-pong mechanism of anion exchange in which the catalytic cycle consists of one pair of exchanging anions per subunit of the band 3 dimer.
已进行了稳态前氯离子外流实验，以直接验证转运抑制剂H2DIDS（4,4'-二异硫氰酸二氢芪-2,2'-二磺酸盐）优先结合转运体向外构象状态的观点。将细胞与含有150 mM磷酸钠、pH 6.2、氮气氛围和80 - 250 μM 36Cl-的培养基平衡。加入H2DIDS（相对于带3为10倍摩尔过量）会诱导氯离子的瞬时外流，这与H2DIDS向外构象的结合比向内构象更紧密的预期一致。H2DIDS诱导的外流大小取决于氯离子浓度，在测试的最高浓度下约为每个细胞700,000个离子。如果阴离子交换的催化循环涉及每个带3二聚体一对交换阴离子，那么瞬时外流的大小会比预期的更大。这些结果与阴离子交换的乒乓机制完全一致，其中催化循环由带3二聚体每个亚基一对交换阴离子组成。
