Differentiating passive from transporter-mediated uptake by PepT1: A comparison and evaluation of four methods

Jeffrey Scow, Srivats Madhavan, Rizwan M. Chaudhry, Ye Zheng, Judith A. Duenes, Michael G. Sarr

Research output: Contribution to journalArticle

5 Citations (Scopus)

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. Km, Vmax, and % gly-sar transported by PepT1 were calculated and compared. Results: Km, Vmax, 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.

Original languageEnglish (US)
Pages (from-to)17-23
Number of pages7
JournalJournal of Surgical Research
Volume170
Issue number1
DOIs
StatePublished - Sep 1 2011

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Sarcosine
Dipeptides
Caco-2 Cells
glycylleucine
Leucine
Protons
Proteins

All Science Journal Classification (ASJC) codes

  • Surgery

Cite this

Scow, Jeffrey ; Madhavan, Srivats ; Chaudhry, Rizwan M. ; Zheng, Ye ; Duenes, Judith A. ; Sarr, Michael G. / Differentiating passive from transporter-mediated uptake by PepT1 : A comparison and evaluation of four methods. In: Journal of Surgical Research. 2011 ; Vol. 170, No. 1. pp. 17-23.
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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. Km, Vmax, and {\%} gly-sar transported by PepT1 were calculated and compared. Results: Km, Vmax, 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.",
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Differentiating passive from transporter-mediated uptake by PepT1 : A comparison and evaluation of four methods. / Scow, Jeffrey; Madhavan, Srivats; Chaudhry, Rizwan M.; Zheng, Ye; Duenes, Judith A.; Sarr, Michael G.

In: Journal of Surgical Research, Vol. 170, No. 1, 01.09.2011, p. 17-23.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Differentiating passive from transporter-mediated uptake by PepT1

T2 - A comparison and evaluation of four methods

AU - Scow, Jeffrey

AU - Madhavan, Srivats

AU - Chaudhry, Rizwan M.

AU - Zheng, Ye

AU - Duenes, Judith A.

AU - Sarr, Michael G.

PY - 2011/9/1

Y1 - 2011/9/1

N2 - 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. Km, Vmax, and % gly-sar transported by PepT1 were calculated and compared. Results: Km, Vmax, 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.

AB - 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. Km, Vmax, and % gly-sar transported by PepT1 were calculated and compared. Results: Km, Vmax, 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.

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