Oligopeptide transport across the basolateral membrane of rat small intestine.

Oligopeptide transport in rat small intestine has been studied in intact tissue, using the luminally and vascularly perfused isolated jejunum in situ technique, and a hydrolysis-resistant dipeptide (D-Phe-L-Gln). The data in this thesis can be divided into two main sections: (l) identification of the transporter proteins, and (2) short-term regulation of transport. The basolateral peptide transporter protein has not, to date, been identified. A candidate protein was identified from membrane vesicles by a photo affinity labelling technique using a dipeptide derivative ([4-azido-3,5-3H-D-Phe]-L-Ala), previously shown to be an efficient substrate for the basolateral transporter. The labelled candidate protein was successfully isolated by 2-DE, which revealed an apparent Mr of 112 ± 2 kDa and a pI of approximately 6.5. Initial sequence analysis, tryptic digestion followed by MALDI-TOF analysis and Q-TOF fragmentation of a tryptic peptide, produced a peptide fingerprint and a sequence tag of 9 amino acids, respectively, which, together, did not completely and conclusively match to any known protein sequence contained within databases, therefore suggesting that the 112 kDa protein may be novel. Short-term regulation of peptide transport was also investigated using the vascular perfusion method. An amino acid-sensing pathway was discovered, using L-Leucine as the regulator, involving protein kinase cacades leading to p70S6k activation and subsequent stimulation ofbasolateral membrane peptide transport. A major conclusion arising from the data was the distinction between PepTl and the basolateral transporter, i.e. the sequence data obtained from the candidate protein did not match to the PepTl sequence; in addition there appeared to be distinct mechanisms of regulatory control at the two membranes. Efficient delivery of peptidomimetic drugs when adminsitered by the oral route requires knowledge of short-term regulation of intestinal peptide transport, in addition to the sequence and structure of the basolateral transporter. This thesis provides essential information, which may eventually contribute to the unequivocal identification and sequencing of the intestinal basolateral peptide transporter, ultimately leading to the future development of compounds with high bioavailability.