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(Biopharmacy)

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概要

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• Human oligopeptide transporter (hPEPT1) translocates di/tri-peptide by coupling to movement of proton down the electrochemical gradient. This transporter has the characteristics that the pH-profile of neutral dipeptide transport shows a bell-shaped curve with an optimal pH of 5.5. In the present study, we examined the reason for the decrease in the acidic region with hPEPT1-transfected CHO cells stably oeverexpressing hPEPT1 (CHO/hPEPT1). The pH profile of the transport activity vs. pH was measured in the presence of nigericin/monensin. Under this condition, the inwardly directed proton concentration gradient was dissipated while the membrane potential remained. As pH increased the activity increased, and the Henderson-Hasselbalch equation with a single pKa was fitted well to the activity curve. The pKa value was estimated to be 6.7±0.2. This value strongly suggests that there is a key amino acid residue, which is involved in pH regulation of transport activity. To identify the key amino acid residue, we examined the effects of various chemical modifications on pH-profile of the transport activity. Modification of carboxyl groups or hydroxyl groups had no significant influence on the pH-profile, whereas a chemical modification of histidine residue with diethylpyrocarbonate (DEPC) completely abolished the transport activity in CHO/hPEPT1 cells. On the other hand, this abolishment was almost prevented by the presence of 10 mM Gly-Sar. This protection was observed only in the presence of the substrate of hPEPT1, indicating that the histidine residue is located at the substrate recognition site. The pH-profile of the transport activity in CHO/hPEPT1 cells treated with DEPC in the presence of 10 mM Gly-Sar also showed a bell-shape similar to that in non-treated CHO/hPEPT1 cells. These data stressed that the histidine residue located at or near the substrate binding site is involved in the pH regulation of transport activity.

• 公益社団法人日本薬学会の論文
• 2006-05-01

著者

• 水谷 忠士

  Faculty Of Advanced Life Science Hokkaido University

• Miyauchi Seiji

  Faculty Of Pharmaceutical Science Matsuyama Univ.

• Kamo Naoki

  Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences

• 水谷 忠士

  Fac. Of Advanced Life Sci. Hokkaido Univ.

• Miyauchi Seiji

  Matsuyama University

• Fujisawa Yuki

  Laboratory Of Biophysical Chemistry Graduate School Of Pharmaceutical Sciences Hokkaido University

• Nara Toshifumi

  College Of Pharmaceutical Sciences Matsuyama Univ

• Kamo Naoki

  Faculty Of Advanced Life Science Hokkaido University

• Kamo Naoki

  Laboratory Of Biophysical Chemistry Faculty Of Advanced Life Science Hokkaido University

• NARA Toshifumi

  Laboratory of Biophysical Chemistry, College of Pharmaceutical Sciences, Matsuyama University

• MIYAUCHI Seiji

  Laboratory of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University

• TATEOKA Ryoko

  Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University

• TAIRA Takahiro

  Molecular Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University

• Kamo Naoki

  Faculty Of Pharmaceutical Sciences Grad. School Of Pharmaceutical Siences Hokkaido Univ.

• Taira Takahiro

  Molecular Biology Graduate School Of Pharmaceutical Sciences Hokkaido University

• Tateoka Ryoko

  Laboratory Of Biophysical Chemistry Graduate School Of Pharmaceutical Sciences Hokkaido University

• Kamo Naoki

  College Of Pharmaceutical Science Matsuyama University Matsuyama Japan

• Miyauchi Seiji

  Laboratory Of Biophysical Chemistry Graduate School Of Pharmaceutical Sciences Hokkaido University

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