Fei Y J, Liu J C, Fujita T, Liang R, Ganapathy V, Leibach F H
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Biochem Biophys Res Commun. 1998 May 8;246(1):39-44. doi: 10.1006/bbrc.1998.8566.
The mammalian peptide transporters PEPT1 and PEPT2 are energized by a transmembrane electrochemical H+ gradient and exhibit similar broad substrate specificity. These transporters however differ in their affinity for substrates, PEPT1 being a low-affinity transporter and PEPT2 being a high-affinity transporter. To identify the substrate binding domain in PEPT1 and PEPT2 which is responsible for the differing affinities, we constructed a series of PEPT1-PEPT2 and PEPT2-PEPT1 chimeras using an in vivo restriction site-independent procedure and determined their substrate affinities. A comparison of these kinetic data for different chimeras with those of the wild-type PEPT1 and PEPT2 in conjunction with the specific structural PEPT1/PEPT2 crossover regions in these chimeras has led to the identification of a putative substrate binding site, which is comprised of the transmembrane domains 7, 8 and 9 of the transporters.
哺乳动物肽转运体PEPT1和PEPT2由跨膜电化学H⁺梯度供能，并表现出相似的广泛底物特异性。然而，这些转运体对底物的亲和力不同，PEPT1是低亲和力转运体，PEPT2是高亲和力转运体。为了鉴定PEPT1和PEPT2中负责不同亲和力的底物结合结构域，我们使用体内不依赖限制性位点的方法构建了一系列PEPT1-PEPT2和PEPT2-PEPT1嵌合体，并测定了它们的底物亲和力。将不同嵌合体的这些动力学数据与野生型PEPT1和PEPT2的动力学数据进行比较，并结合这些嵌合体中特定的结构PEPT1/PEPT2交叉区域，从而鉴定出一个推定的底物结合位点，该位点由转运体的跨膜结构域7、8和9组成。
