Salhany J M, Rauenbuehler P B, Sloan R L
Veterans Administration Medical Center, Omaha, Nebraska 68105.
J Biol Chem. 1987 Nov 25;262(33):15974-8.
When pyridoxal 5'-phosphate (PLP) is covalently bound to band 3 protein in intact red blood cells and those cells are subjected to the osmotic hemolysis and resealing process, a significant reduction in the original PLP anion transport inhibitory potency occurs. We show that partial deinhibition is not due to the development of a second anion transport pathway in resealed ghosts. Rather, partial deinhibition arises from a hemolysis-induced conformational change in CH17 (17-kDa integral chymotryptic domain of band 3). This change causes the extracellular exposure of new transport inhibitory sites. Exposure of the new sites leads to a 2-fold increase in PLP labeling of CH17 in resealed ghosts compared with CH17 in intact red cells. The hemolysis and resealing process has no effect on the labeling of CH35 (35-kDa integral chymotryptic fragment of band 3). Double-labeling studies show restoration of transport inhibitory potency to near red cell levels when the newly exposed CH17 sites are labeled with PLP in resealed ghosts. The results support the view that CH17 contains PLP transport inhibitory sites. They show that a major conformational change occurs in band 3 with hemolysis.
当吡哆醛5'-磷酸（PLP）与完整红细胞中的带3蛋白共价结合，且这些细胞经历渗透性溶血和重新封闭过程时，原始PLP对阴离子转运的抑制效力会显著降低。我们发现部分去抑制并非由于重新封闭的血影中出现了第二条阴离子转运途径。相反，部分去抑制源于溶血诱导的带3蛋白CH17（17 kDa完整胰凝乳蛋白酶结构域）构象变化。这种变化导致新的转运抑制位点暴露于细胞外。与完整红细胞中的CH17相比，新位点的暴露使重新封闭血影中CH17的PLP标记增加了2倍。溶血和重新封闭过程对带3蛋白的CH35（35 kDa完整胰凝乳蛋白酶片段）标记没有影响。双标记研究表明，当重新封闭血影中新暴露的CH17位点用PLP标记时，转运抑制效力恢复到接近红细胞水平。这些结果支持了CH17含有PLP转运抑制位点的观点。它们表明带3蛋白在溶血时会发生重大构象变化。
