Iseki K, Naasani I, Kikuchi T, Sugawara M, Kobayashi M, Kohri N, Miyazaki K
Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Sapporo, Japan.
Biochim Biophys Acta. 1998 Jan 19;1368(2):329-37. doi: 10.1016/s0005-2736(97)00201-0.
The carrier protein(s) responsible for the transport of ceftibuten, a peptide-like dianionic cefem, in rat renal brush-border membrane were solubilized and purified by a ceftibuten-ligand specific affinity chromatography technique. The proteoliposomes reconstituted from the solubilized brush-border membrane proteins by dialysis had H+-sensitive uptake of ceftibuten and trans-stimulative effect by cephalexin. A specific uptake activity for ceftibuten was found in the 3.5 M-eluted fraction but not the flowthrough and the 0.5 M-eluted fraction of the affinity chromatography. Analyzing this active fraction by SDS/PAGE after reconstituting into liposomes gave two major proteins (approx. molecular masses of 130 and 107 kDa). The purification protocol presented in this study permitted an efficient isolation of the carrier proteins responsible for the transport of ceftibuten and other peptide-like compounds.
通过头孢布烯配体特异性亲和色谱技术，溶解并纯化了负责在大鼠肾刷状缘膜中转运头孢布烯（一种肽样二阴离子头孢菌素）的载体蛋白。通过透析从溶解的刷状缘膜蛋白中重构的蛋白脂质体对头孢布烯具有H⁺敏感性摄取，并且受到头孢氨苄的反刺激作用。在亲和色谱的3.5M洗脱级分中发现了对头孢布烯的特异性摄取活性，但在流出液和0.5M洗脱级分中未发现。将该活性级分重构到脂质体中后通过SDS/PAGE分析得到两种主要蛋白质（分子量约为130和107kDa）。本研究中提出的纯化方案允许有效分离负责头孢布烯和其他肽样化合物转运的载体蛋白。
