Model Analysis of the Apparent Saturation Kinetics of Purine Nucleobase Uptake in Cells co-Expressing Transporter and Metabolic Enzyme

Abstract

Purpose: This study aims to understand the effect of salvage enzyme activity on the saturable kinetics of facilitated cellular uptake of purine nucleobase by developing a cellular kinetic model incorporating equilibrative nucleobase transporter 1 (ENBT1) and adenine phosphoribosyltransferase (APRT), with adenine as a model nucleobase.

Methods: A cellular kinetic model incorporating the functions of ENBT1 and APRT was developed using Napp software and employed for model-based analysis of the cellular disposition of adenine.

Results: Simulation analysis using the developed cellular kinetic model could account for the experimentally observed time-dependent changes in the K_m(app) value of adenine for ENBT1-mediated uptake. At a long experimental time, the model shows that uptake of adenine is rate-limited by APRT, enabling determination of the K_m value for APRT. At early time, the rate-limiting step for adenine uptake is ENBT1-mediated transport, enabling determination of the K_m value for ENBT1. Further simulations showed that the effect of experimental time on the K_m(app) value for ENBT1-mediated uptake is dependent on the APRT expression level.

Conclusion: Our findings indicate that both enzyme expression levels and experimental time should be considered when using cellular uptake studies to determine the K_m values of purine nucleobases for facilitated transporters.

Keywords: Adenine; Equilibrative nucleobase transporter 1 (ENBT1); adenine phosphoribosyltransferase (APRT); modeling & simulation (M&S); uptake study.