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.

中文翻译：

---

共表达转运蛋白和代谢酶的细胞中嘌呤核碱基摄取的表观饱和动力学模型分析

目的

本研究旨在通过建立包含平衡核碱基转运蛋白 1 (ENBT1) 和腺嘌呤磷酸核糖基转移酶 (APRT) 的细胞动力学模型，以腺嘌呤作为模型核碱基，了解补救酶活性对促进细胞摄取嘌呤核碱基的饱和动力学的影响。

方法

使用 Napp 软件开发了一个包含 ENBT1 和 APRT 功能的细胞动力学模型，并用于基于模型的腺嘌呤细胞分布分析。

结果

使用开发的细胞动力学模型进行的模拟分析可以解释实验观察到的腺嘌呤K _m(app)值对 ENBT1 介导的摄取的时间依赖性变化。在较长的实验时间内，该模型显示腺嘌呤的摄取受到 APRT 的速率限制，从而能够确定 APRT 的K _m值。在早期，腺嘌呤摄取的限速步骤是 ENBT1 介导的转运，从而能够确定 ENBT1 的K _m值。进一步的模拟表明，实验时间对 ENBT1 介导摄取的K _m(app)值的影响取决于 APRT 表达水平。

结论

我们的研究结果表明，在使用细胞摄取研究来确定促进转运蛋白的嘌呤核碱基的K _{m值时，应同时考虑酶表达水平和实验时间。}