Tiruppathi C, Kulanthaivel P, Ganapathy V, Leibach F H
Department of Cell and Molecular Biology, Medical College of Georgia, Augusta 30912-2100.
Biochem J. 1990 May 15;268(1):27-33. doi: 10.1042/bj2680027.
L-Phe-L-Pro-L-Ala is a tripeptide which is hydrolysable almost exclusively by dipeptidyl peptidase IV in rabbit renal brush-border membrane vesicles. In order to delineate the mechanism of the transport of an intact tripeptide across the brush-border membrane, we studied the characteristics of the uptake of [3H]Phe-Pro-Ala in membrane vesicles in which the activity of dipeptidylpeptidase IV was completely inhibited by treatment with di-isopropyl fluorophosphate. In these vesicles, uptake of radiolabel from the tripeptide was found to be Na(+)-independent, but was greatly stimulated by an inwardly directed H+ gradient. The H(+)-gradient-dependent radiolabel uptake appeared to be an active process, because the time course of uptake exhibited an overshoot phenomenon. The process was also electrogenic, being stimulated by an inside-negative membrane potential. Under the uptake-measurement conditions there was no detectable hydrolysis of [3H]Phe-Pro-Ala in the incubation medium when di-isopropyl fluorophosphate-treated membrane vesicles were used. Analysis of intravesicular contents revealed that the radiolabel inside the vesicles was predominantly (greater than 90%) in the form of intact tripeptide. These data indicate that the uptake of radiolabel from [3H]Phe-Pro-Ala in the presence of an inwardly directed H+ gradient represents almost exclusively uptake of intact tripeptide. Uphill transport of the tripeptide was also demonstrable in the presence of an inwardly directed Na+ or K+ gradient, but only if nigericin was added to the medium. Under these conditions, nigericin, an ionophore for Na+, K+ and H+, was expected to generate a transmembrane H+ gradient. Uptake of Phe-Pro-Ala in the presence of a H+ gradient was inhibited by di- and tri-peptides, but not by free amino acids. It is concluded that tripeptide/H+ co-transport is the mechanism of Phe-Pro-Ala uptake in rabbit renal brush-border membrane vesicles.
L-苯丙氨酸-L-脯氨酸-L-丙氨酸是一种三肽，在兔肾刷状缘膜囊泡中几乎只能被二肽基肽酶IV水解。为了阐明完整三肽跨刷状缘膜的转运机制，我们研究了在经二异丙基氟磷酸处理使二肽基肽酶IV活性完全被抑制的膜囊泡中[3H]苯丙氨酸-脯氨酸-丙氨酸的摄取特性。在这些囊泡中，发现从三肽摄取放射性标记物是不依赖Na(+)的，但受到内向H+梯度的极大刺激。H(+)梯度依赖性放射性标记物摄取似乎是一个主动过程，因为摄取的时间进程表现出过冲现象。该过程也是生电的，受到内膜负电位的刺激。在摄取测量条件下，当使用经二异丙基氟磷酸处理的膜囊泡时，孵育培养基中未检测到[3H]苯丙氨酸-脯氨酸-丙氨酸的水解。对囊泡内成分的分析表明，囊泡内的放射性标记物主要（超过90%）以完整三肽的形式存在。这些数据表明，在存在内向H+梯度的情况下，从[3H]苯丙氨酸-脯氨酸-丙氨酸摄取放射性标记物几乎完全代表完整三肽的摄取。在存在内向Na+或K+梯度的情况下，三肽的上坡转运也得到了证实，但前提是向培养基中加入尼日利亚菌素。在这些条件下，尼日利亚菌素是一种Na+、K+和H+的离子载体，预计会产生跨膜H+梯度。在存在H+梯度的情况下，苯丙氨酸-脯氨酸-丙氨酸的摄取受到二肽和三肽的抑制，但不受游离氨基酸的抑制。结论是，三肽/H+共转运是兔肾刷状缘膜囊泡中苯丙氨酸-脯氨酸-丙氨酸摄取的机制。
