Modeling of Cellular Arginine Uptake by More Than One Transporter

Modeling of Cellular Arginine Uptake by More Than One Transporter Determining the kinetic constants of arginine uptake by endothelial cells mediated by more than one transporter from linearization of data as Eadie-Hofstee plots or modeling which does not include the concentration of trace radiolabeled amino acid used to measure uptake may not be correct. The initial rate of uptake of trace [3H]l-arginine by HUVECs and ECV304 cells in the presence of a range of unlabeled arginine and modifiers was used in nonlinear models to calculate the constants of arginine uptake using GraphPad Prism. Theoretical plots of uptake derived from constants determined from Eadie-Hofstee graphs overestimated uptake, whereas those from the nonlinear modeling approach agreed with experimental data. The contribution of uptake by individual transporters could be modeled and showed that leucine inhibited the individual transporters differently and not necessarily competitively. N-Ethylmaleimide inhibited only y+ transport, and BCH may be a selective inhibitor of y+L transport. The absence of sodium reduced arginine uptake by y+L transport and reduced the K m′, whereas reducing sodium decreased arginine uptake by y+ transport without affecting the K m′. The nonlinear modeling approach using raw data avoided the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. This study provides explanations for discrepancies in the literature and suggests that a nonlinear modeling approach better characterizes the kinetics of amino acid uptake into cells by more than one transporter. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Modeling of Cellular Arginine Uptake by More Than One Transporter

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Publisher
Springer-Verlag
Copyright
Copyright © 2011 by Springer Science+Business Media, LLC
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-011-9408-0
Publisher site
See Article on Publisher Site

Abstract

Determining the kinetic constants of arginine uptake by endothelial cells mediated by more than one transporter from linearization of data as Eadie-Hofstee plots or modeling which does not include the concentration of trace radiolabeled amino acid used to measure uptake may not be correct. The initial rate of uptake of trace [3H]l-arginine by HUVECs and ECV304 cells in the presence of a range of unlabeled arginine and modifiers was used in nonlinear models to calculate the constants of arginine uptake using GraphPad Prism. Theoretical plots of uptake derived from constants determined from Eadie-Hofstee graphs overestimated uptake, whereas those from the nonlinear modeling approach agreed with experimental data. The contribution of uptake by individual transporters could be modeled and showed that leucine inhibited the individual transporters differently and not necessarily competitively. N-Ethylmaleimide inhibited only y+ transport, and BCH may be a selective inhibitor of y+L transport. The absence of sodium reduced arginine uptake by y+L transport and reduced the K m′, whereas reducing sodium decreased arginine uptake by y+ transport without affecting the K m′. The nonlinear modeling approach using raw data avoided the errors inherent in methods deriving constants from the linearization of the uptake processes following Michaelian kinetics. This study provides explanations for discrepancies in the literature and suggests that a nonlinear modeling approach better characterizes the kinetics of amino acid uptake into cells by more than one transporter.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Nov 24, 2011

References

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