title: Differentiating passive from transporter-mediated uptake by PepT1: a comparison and evaluation of four methods.
abstract: BACKGROUND
  To quantify transmembrane transport of dipeptides by PepT1, passive uptake (non-PepT1 mediated) must be subtracted from total (measured) uptake. Three methods have been described to estimate passive uptake: perform experiments at cold temperatures, inhibit target dipeptide uptake with a greater concentration of a second dipeptide, or use modified Michaelis-Menten kinetics. We hypothesized that performing uptake experiments at pH 8.0 would estimate passive uptake accurately, because PepT1 requires a proton gradient. Our aim was to determine the most accurate method to estimate passive uptake.
  
  
  METHODS
  Caco-2 cells were incubated with various concentrations of glycyl-sarcosine (gly-sar) at pH 6.0 and at 37°C to measure total uptake. Passive uptake was estimated: (1) by incubating Caco-2 cells with varying concentrations of gly-sar at 4°C, (2) in the presence of 50 mM glycyl-leucine, (3) in solution at pH 8.0, or (4) using modified Michaelis-Menten kinetics. PepT1-mediated uptake was calculated by subtracting passive uptake from total uptake. K(m), V(max), and % gly-sar transported by PepT1 were calculated and compared.
  
  
  RESULTS
  K(m), V(max), and % gly-sar transported by PepT1 varied from 0.7 to 2.4 mM, 8.4 to 21.0 nmol/mg protein/10 min, and 69% to 87%, respectively. Uptakes calculated with cold, 50 mM gly-leu and using modified Michaelis-Menten kinetics were similar but differed significantly from uptake at pH 8.0 (P < 0.001).
  
  
  CONCLUSIONS
  Estimating passive uptake at pH 8.0 does not appear to be accurate. Measuring uptake at cold temperatures or in the presence of a greater concentration of a second dipeptide, and confirming results with modified Michaelis-Menten kinetics is recommended.
authors.affiliation: Department of Surgery and GI Research Unit, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Scow
authors.forename: Srivats
authors.initials: S
authors.lastname: Madhavan
authors.forename: Rizwan M
authors.initials: RM
authors.lastname: Chaudhry
authors.forename: Ye
authors.initials: Y
authors.lastname: Zheng
authors.forename: Judith A
authors.initials: JA
authors.lastname: Duenes
authors.forename: Michael G
authors.initials: MG
authors.lastname: Sarr
mesh_terms: D001692:Biological Transport; D018938:Caco-2 Cells; D004151:Dipeptides / Q000493:pharmacokinetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D000074011:Peptide Transporter 1; D027981:Symporters / Q000502:physiology*
chemical_list: D004151:Dipeptides; D000074011:Peptide Transporter 1; C082186:SLC15A1 protein, human; D027981:Symporters; C004194:glycylsarcosine