Naasani I, Kikuchi T, Sugawara M, Kobayashi M, Iseki K, Miyazaki K
Department of Pharmacy, Hokkaido University Hospital, School of Medicine, Hokkaido University, Sapporo, Japan.
Biochim Biophys Acta. 1996 Sep 4;1283(2):185-91. doi: 10.1016/0005-2736(96)00092-2.
Optimal procedures for the reconstitution of the transport activity of ceftibuten, a dianionic beta-lactam antibiotic, from rat kidney brush-border membrane were developed. The uptake activity into reconstituted proteoliposomes appeared to be particularly sensitive to the extraction conditions, and to the lipid composition used for reconstitution. Changes in the concentration of octyl glucoside significantly affected the extraction of ceftibuten transport activity, and optimal extraction was achieved at a concentration of 60 mM. Optimal reconstitution was achieved using a lipid composition of asolectin, cholesterol and phosphatidylserine in a w/w percent ratio of 60:30:10, respectively, and with a lipid-to-protein ratio of 10. The uptake of ceftibuten into the resulting proteoliposomes showed temperature and pH dependency, was inhibitable by a range of cephem antibiotics, oligopeptides and the organic anion PAH, and was trans-stimulated by cephalexin and dipeptides. This reconstitution system will likely prove useful in future studies on the functional analysis of the peptide transport system in a purified form.
已开发出用于重构大鼠肾刷状缘膜中双阴离子β-内酰胺抗生素头孢布烯转运活性的最佳方法。重构的蛋白脂质体对头孢布烯的摄取活性似乎对提取条件以及用于重构的脂质组成特别敏感。辛基葡糖苷浓度的变化显著影响头孢布烯转运活性的提取，在60 mM浓度下实现了最佳提取。使用大豆卵磷脂、胆固醇和磷脂酰丝氨酸的脂质组合物，其重量比分别为60:30:10，脂质与蛋白质的比例为10，实现了最佳重构。所得蛋白脂质体对头孢布烯的摄取表现出温度和pH依赖性，可被一系列头孢菌素抗生素、寡肽和有机阴离子对氨基马尿酸抑制，并受到头孢氨苄和二肽的反刺激。该重构系统可能在未来以纯化形式对肽转运系统进行功能分析的研究中证明是有用的。
