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

Alghamdi, Othman A; King, Nicola; Jones, Graham L; Moens, Pierre

doi:10.1016/j.bbamem.2017.12.021

Author(s)

Alghamdi, Othman A

King, Nicola

Jones, Graham L

Moens, Pierre

Publication Date

2018

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.

Citation

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1860(5), p. 960-964

ISSN

0005-2736

Link

https://hdl.handle.net/1959.11/23159

Language

en

Publisher

Elsevier BV

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

Type of document

Journal Article

Entity Type

Publication

Author(s)	Alghamdi, Othman A King, Nicola Jones, Graham L Moens, Pierre
Publication Date	2018
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.
Citation	Biochimica et Biophysica Acta (BBA) - Biomembranes, 1860(5), p. 960-964
ISSN	0005-2736
Link	https://hdl.handle.net/1959.11/23159
Language	en
Publisher	Elsevier BV
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
Type of document	Journal Article
Entity Type	Publication

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

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