Abstract
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.