Please use this identifier to cite or link to this item:
Title: A new use of β-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla
Contributor(s): Alghamdi, Othman A (author); King, Nicola (author); Jones, Graham L  (author)orcid ; Moens, Pierre  (author)orcid 
Publication Date: 2018
DOI: 10.1016/j.bbamem.2017.12.021
Handle Link:
Abstract: Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered diand tripeptides alongside a wide range of peptidomimetic drugs in the kidney. The aim of this study was to investigate the potential use of the fluorophore-conjugated dipeptide β-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMVOC) and outer medulla (BBMV-OM) (representing PEPT1 and PEPT2 respectively). The vesicles were isolated using a dual magnesium precipitation and centrifugation technique. Intravesicular fluorescence accumulation was measured after incubating extra-vesicular media at pH 6.6 and different concentrations of β-Ala-Lys (AMCA) with vesicles pre-equilibrated at pH 7.4. Both BBMV-OC and BMMV-OM showed accumulation of an intravesicular fluorescence signal after 20 min incubation. Changing the extra-vesicular pH to 7.4 caused a significant reduction in the β-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations> 100 μM. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100 μM β-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of β-Ala-Lys (AMCA) at 20 min showed that the Km and Vmax were 783.7 ± 115.7 μM and 2191.2 ± 133.9 ΔF/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at Km = 93.6 ± 21.9 μM and Vmax = 935.8 ± 50.2 ΔF/min/mg. These findings demonstrate the applicability of β-Ala-Lys (AMCA) as a biosensor to measure the transport activity of the renal-type PEPT1 and PEPT2 in BBMV-OC and BMMV-OM respectively.
Publication Type: Journal Article
Source of Publication: Biochimica et Biophysica Acta (BBA) - Biomembranes, 1860(5), p. 960-964
Publisher: Elsevier BV
Place of Publication: The Netherlands
ISSN: 0005-2736
Field of Research (FOR): 111602 Human Biophysics
111601 Cell Physiology
060110 Receptors and Membrane Biology
Socio-Economic Outcome Codes: 970106 Expanding Knowledge in the Biological Sciences
Peer Reviewed: Yes
HERDC Category Description: C1 Refereed Article in a Scholarly Journal
Statistics to Oct 2018: Visitors: 6
Views: 27
Downloads: 0
Appears in Collections:Journal Article
School of Science and Technology

Files in This Item:
2 files
File Description SizeFormat 
Show full item record

Page view(s)

checked on May 2, 2019
Google Media

Google ScholarTM



Items in Research UNE are protected by copyright, with all rights reserved, unless otherwise indicated.