Tamai I, Tomizawa N, Kadowaki A, Terasaki T, Nakayama K, Higashida H, Tsuji A
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kanazawa University, Japan.
Biochem Pharmacol. 1994 Aug 30;48(5):881-8. doi: 10.1016/0006-2952(94)90358-1.
An intestinal active transport system specific to small peptides and peptide-like drugs such as beta-lactam antibiotics was functionally expressed in Xenopus laevis oocytes after microinjection of messenger RNA (mRNA) derived from rat intestinal mucosal cells. The transport activity was evaluated by measuring the uptake of a tripeptide-like cephalosporin antibiotic, ceftibuten, which has high affinity for the intestinal peptide/H+ co-transporter and is resistant to peptidases. Ceftibuten transport in mRNA-injected oocytes was pH dependent (a proton gradient is the driving force), stereo selective (uptake of the cis-isomer of ceftibuten was about 4-fold higher than that of the trans-isomer), saturable and temperature dependent. Furthermore, various dipeptides showed cis-inhibitory and trans-stimulatory effects on the uptake of ceftibuten by mRNA-injected oocytes, suggesting that ceftibuten and dipeptides are transported by a common carrier protein. These results are in accordance with the functional properties of native proton-coupled peptide transporter previously clarified by studies with isolated intestinal brush-border membrane vesicles and other experimental systems. A protein with a molecular mass of about 130 kDa expressed in the membrane of mRNA-injected oocytes was identified as the transport protein by specific labeling with a photoreactive beta-lactam antibiotic, [3H]benzylpenicillin, followed by SDS-PAGE analysis of the radiolabeled protein. Furthermore, an experiment with mRNA size-fractionated by sucrose density gradient centrifugation indicated that the peptide transporter is encoded by mRNA of between 1.8 and 3.6 kb. These results, obtained using a heterologous gene expression technique, confirm that intestinal absorption of beta-lactam antibiotics occurs through a carrier-mediated mechanism and show that biologically stable beta-lactam antibiotics can be useful probes for molecular analysis of intestinal peptide transporter.
在将源自大鼠肠黏膜细胞的信使核糖核酸（mRNA）显微注射到非洲爪蟾卵母细胞后，一种对小肽和肽类药物（如β-内酰胺抗生素）具有特异性的肠道主动转运系统在其中功能性表达。通过测量三肽样头孢菌素抗生素头孢布烯的摄取来评估转运活性，头孢布烯对肠道肽/H⁺共转运体具有高亲和力且对肽酶具有抗性。注射mRNA的卵母细胞中头孢布烯的转运是pH依赖性的（质子梯度是驱动力）、立体选择性的（头孢布烯顺式异构体的摄取比反式异构体高约4倍）、可饱和的且温度依赖性的。此外，各种二肽对注射mRNA的卵母细胞摄取头孢布烯表现出顺式抑制和反式刺激作用，这表明头孢布烯和二肽由共同的载体蛋白转运。这些结果与先前通过分离的肠刷状缘膜囊泡和其他实验系统研究阐明的天然质子偶联肽转运体的功能特性一致。通过用光反应性β-内酰胺抗生素[³H]苄青霉素进行特异性标记，然后对放射性标记蛋白进行SDS-PAGE分析，在注射mRNA的卵母细胞膜中表达的一种分子量约为130 kDa的蛋白质被鉴定为转运蛋白。此外，一项用蔗糖密度梯度离心法对mRNA进行大小分级的实验表明，肽转运体由1.8至3.6 kb的mRNA编码。这些使用异源基因表达技术获得的结果证实，β-内酰胺抗生素的肠道吸收是通过载体介导的机制发生的，并表明生物稳定的β-内酰胺抗生素可作为肠道肽转运体分子分析的有用探针。
