2006 Volume 29 Issue 5 Pages 997-1005
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.