Please use this identifier to cite or link to this item: https://hdl.handle.net/1959.11/23159
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: https://hdl.handle.net/1959.11/23159
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
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Appears in Collections:Journal Article
School of Science and Technology

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