Identification of the histidine residues involved in substrate recognition by a rat H<sup>+</sup>/peptide cotransporter, PEPT1

Takato, Tsuyoshi; Saya, Hideyuki; Mukai, Mayumi; Inui, Ken Ichi

doi:10.1016/0014-5793(96)00952-0

Cited by 83 publications

(48 citation statements)

References 28 publications

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“…Up to date, histidyl residues have been reported extensively to be one of the most important amino acid residues in hPEPT1, which were revealed by DEPC modification 26,27,35,36) and site-directed mutagenesis studies. [28][29][30]37) DEPC modification showed non-competitive inhibition 26,27) and protection in the presence of substrates. 35,36) Site-directed mutagenesis studies revealed that His-57 in the second transmembrane domain (TMD2) is the most likely residue involved in the H ϩ binding/dissociation.…”

Section: Discussionmentioning

confidence: 97%

“…35,36) Site-directed mutagenesis studies revealed that His-57 in the second transmembrane domain (TMD2) is the most likely residue involved in the H ϩ binding/dissociation. [27][28][29][30]37) The site-directed mutagenesis studies affecting His-121 in TMD4 showed that it is the substrate-binding site. 28) His-57 and His-121 were hypothesized to be intimately located and form the substrate binding pockets.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, histidine residues in PEPT1 have been identified as key amino acid residues, [26][27][28][29][30] which are involved in substrate recognition as well as responsible for the transport activity, especially proton-translocation. Therefore, we checked the effect of chemical modification of histidine by DEPC on the pH profile of transport activity.…”

Section: Fig 5 Ph Dependency Of Gly-sar Transport Activities When Omentioning

confidence: 99%

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The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Fujisawa

Tateoka

Nara

et al. 2006

Biological & Pharmaceutical Bulletin

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It has been estimated that more than 80% of amino acids found in the intestinal lumen after a protein meal are in the form of small peptides rather than free amino acids.1) Therefore, a majority of protein digestion products are obligatorily absorbed via an intestine oligo-peptide transporter called PEPT1. Of interest, PEPT1 also mediates the absorption of a huge number of pharmacologically important compounds including peptidomimetic drugs, 2,3) such as oral b-lactam antibiotics, 4-6) ACE-inhibitors like captopril, 7) or the peptidase inhibitor bestatine 8) and even compounds without an obvious peptide bond or equivalent, 9) such as d-amino levulinic acid.10) This surprisingly high tolerance for structural diversity of the substrate by PEPT1 has been expected to be used as a channel to increase the intestinal absorption of poorly absorbable drugs. 11)The transport of substrates via PEPT1 is coupled to the downward movement of a proton in accordance with its electrochemical gradient. 2,3,12) It has been demonstrated in intestinal membrane vesicles, 4,13,14) Xenopus oocytes 15) and culture cells 16) that the transport activity of PEPT1 shows a bellshaped pH dependence with an optimal pH 5.5 to 6.0. This optimal pH is physiologically collaborated with an acidic unstirred water layer (pH 5.5-6.0) in the intestinal lumen. 1,17) Hence, PEPT1 can function most efficiently in epithelial cells of the small intestine.As for the bell-shaped activity of PEPT1 vs. pH, the reason why the transport activity decreases with an increase in pH is obviously the decrease in the driving force of the proton electrochemical potential gradient. On the other hand, why does the activity decrease in a more acidic region although the driving force should increase? To answer this question, we will try to obtain a relationship between the transport activity and extracellular pH. However, there is a problem to be solved: The driving force changes along with the change in extracellular pH. The pH-dependent properties of PEPT1 should be determined without changing the driving force. Here, we employ the experimental condition that the proton concentration gradient across the membrane is dissipated by the addition of monensin and nigericin 18,19) ; then the driving force is solely the membrane potential. To perform this experiment, a cell line is necessary which shows high transport activity. First, we established stably transfected CHO cells which show high transport activities for various substrates with almost the same K m values as those obtained from those of a model human intestine epithelial cell line, Caco-2. [20][21][22] Thus, this CHO cell line over-expressing hPEPT1 (designated as CHO/hPEPT1) is useful and convenient for transport studies of hPEPT1. In the present study, we then examined the pH-dependency of transport activity by hPEPT1 using CHO/hPEPT1 cells. Human oligopeptide transporter (hPEPT1) translocates di/tri-peptide by coupling to movement of proton down the electrochemical gradient. This transporter has the characteristics ...

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Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Fig 5 Ph Dependency Of Gly-sar Transport Activities When Omentioning

confidence: 99%

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The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Fujisawa

Tateoka

Nara

et al. 2006

Biological & Pharmaceutical Bulletin

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“…Cell lysate was directly prepared from the cells with 1% NP-40, and crude membrane fractions were prepared as described previously, with some modifications (42). Briefly, cells were homogenated by sonication in the buffer (250 mM sucrose and 5 mM HEPES, pH 7.4) and were then centrifuged (2,000 g, 10 min).…”

Section: Methodsmentioning

confidence: 99%

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Yonezawa¹,

Masuda²,

Katsura³

et al. 2008

American Journal of Physiology-Cell Physiology

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101

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“…Further studies are necessary to assess the structural/functional significance of this stretch. Other regions/residues that are recognized as structurally or functionally I. Rønnestad and others Terada et al, 1996) Amino acid notation follows the single letter code.…”

Section: Fish Vs Tetrapod Pept1 Proteins: a Comparisonmentioning

confidence: 99%

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhuaL.): cloning, tissue expression and comparative aspects

Rønnestad

Gavaia

Viegas

et al. 2007

Journal of Experimental Biology

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A novel full-length cDNA that encodes for the Atlantic cod (Gadus morhua L.) PepT1-type oligopeptide transporter has been cloned. This cDNA (named codPepT1) was 2838·bp long, with an open reading frame of 2190·bp encoding a putative protein of 729 amino acids. Comparison of the predicted Atlantic cod PepT1 protein with zebrafish, bird and mammalian orthologs allowed detection of many structural features that are highly conserved among all the vertebrate proteins analysed, including (1) a larger than expected area of hydrophobic amino acids in close proximity to the N terminus; (2) a single highly conserved cAMP/cGMP-dependent protein kinase phosphorylation motif; (3) a large N-glycosylationrich region within the large extracellular loop; and (4) a conserved and previously undescribed stretch of 8-12 amino acid residues within the large extracellular loop. Expression analysis at the mRNA level indicated that Atlantic cod PepT1 is mainly expressed at intestinal level, but that it is also present in kidney and spleen. Analysis of its regional distribution along the intestinal tract of the fish revealed that PepT1 is ubiquitously expressed in all segments beyond the stomach, including the pyloric caeca, and through the whole midgut. Only in the last segment, which included the hindgut, was there a lower expression. Atlantic cod PepT1, the second teleost fish PepT1-type transporter documented to date, will contribute to the elucidation of the evolutionary and functional relationships among vertebrate peptide transporters. Moreover, it can represent a useful tool for the study of gut functional regionalization, as well as a marker for the analysis of temporal and spatial expression during ontogeny.

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Identification of the histidine residues involved in substrate recognition by a rat H⁺/peptide cotransporter, PEPT1

Cited by 83 publications

References 28 publications

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhuaL.): cloning, tissue expression and comparative aspects

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Identification of the histidine residues involved in substrate recognition by a rat H+/peptide cotransporter, PEPT1

Cited by 83 publications

References 28 publications

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

The Extracellular pH Dependency of Transport Activity by Human Oligopeptide Transporter 1 (hPEPT1) Expressed Stably in Chinese Hamster Ovary (CHO) Cells: A Reason for the Bell-Shaped Activity versus pH

Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

Oligopeptide transporter PepT1 in Atlantic cod (Gadus morhuaL.): cloning, tissue expression and comparative aspects

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Identification of the histidine residues involved in substrate recognition by a rat H⁺/peptide cotransporter, PEPT1