Verri T, Maffia M, Danieli A, Herget M, Wenzel U, Daniel H, Storelli C
Laboratory of General Physiology, Department of Biology, University of Lecce, Strada Provinciale Lecce-Monteroni, I-73100 Lecce, Italy.
J Exp Biol. 2000 Oct;203(Pt 19):2991-3001. doi: 10.1242/jeb.203.19.2991.
H(+)/peptide cotransport in brush-border membrane vesicles (BBMVs) from eel (Anguilla anguilla) intestine was studied by measuring d-[(3)H]-phenylalanyl-l-alanine uptake and by monitoring peptide-dependent intravesicular acidification using the pH-sensitive dye Acridine Orange. d-[(3)H]-phenylalanyl-l-alanine influx was greatly stimulated by an inside-negative membrane potential and enhanced by an inwardly directed H(+) gradient. In parallel, vesicular H(+) influx was significantly increased in the presence of extravesicular d-phenylalanyl-l-alanine or a series of glycyl and l-prolyl peptides. H(+)/peptide cotransport displayed saturable kinetics involving a single carrier system with apparent substrate affinities of 0.9-2.6 mmol l(-1) depending on the particular peptide. All substrates tested competed with this system. Pre-incubation of BBMVs with dipeptides prevented diethylpyrocarbonate inhibition of transport activity, suggesting that the substrates mask histidine residues involved in the catalytic function of the transporter. Using human PepT1-specific primers, a reverse transcription-polymerase chain reaction (RT-PCR) signal was detected in eel intestine. Our results suggest that, in eel intestine, a brush-border membrane 'low-affinity'-type H(+)/peptide cotransport system is present that shares kinetic features with the mammalian intestinal PepT1-type transporters.
通过测量d-[(3)H]-苯丙氨酰-L-丙氨酸摄取，并使用pH敏感染料吖啶橙监测肽依赖性囊泡内酸化，研究了鳗鱼（欧洲鳗鲡）肠道刷状缘膜囊泡（BBMVs）中的H(+)/肽共转运。内向负膜电位极大地刺激了d-[(3)H]-苯丙氨酰-L-丙氨酸的流入，并因内向的H(+)梯度而增强。同时，在囊泡外存在d-苯丙氨酰-L-丙氨酸或一系列甘氨酰和L-脯氨酰肽时，囊泡内的H(+)流入显著增加。H(+)/肽共转运表现出饱和动力学，涉及单一载体系统，根据特定肽的不同，其表观底物亲和力为0.9 - 2.6 mmol l(-1)。所有测试的底物都与该系统竞争。用二肽预孵育BBMVs可防止焦碳酸二乙酯对转运活性的抑制，这表明底物掩盖了参与转运体催化功能的组氨酸残基。使用人PepT1特异性引物，在鳗鱼肠道中检测到逆转录-聚合酶链反应（RT-PCR）信号。我们的结果表明，在鳗鱼肠道中存在一种刷状缘膜“低亲和力”型H(+)/肽共转运系统，它与哺乳动物肠道PepT1型转运体具有共同的动力学特征。
