A new use of beta-Ala-Lys (AMCA) as a transport reporter for PEPT1 and PEPT2 in renal brush border membrane vesicles from the outer cortex and outer medulla

摘要

Integral membrane proteins PEPT1 and PEPT2 are essential for reabsorbing almost all hydrolysed or filtered di- and 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 beta-Ala-Lys (AMCA) as a biosensor for measuring peptide transport activity in brush border membrane vesicles isolated from the outer cortex (BBMV-OC) 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 beta-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 beta-Ala-Lys (AMCA) uptake into BBMV-OC at concentrations 100 mu M. When different concentrations of dipeptide, Gly-Gln was added, there was a significant inhibition of 100 mu M beta-Ala-Lys (AMCA) uptake into BBMV-OC and BMMV-OM, reaching 69% and 80%, respectively. Kinetic analysis of beta-Ala-Lys (AMCA) at 20 min showed that the K-m and V-max were 783.7 +/- 115.7 mu M and 2191.2 +/- 133.9 Delta F/min/mg for BBMV-OC, while BMMV-OM showed significantly higher affinity, but lower capacity at K-m = 93.6 +/- 21.9 mu M and V-max = 935.8 +/- 50.2 Delta F/min/mg. These findings demonstrate the applicability of beta-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.