Previous work demonstrated that human liver microsomes (HLMs) can spontaneously bind to silica-coated magnetizable beads (HLM-beads) and that these HLM-beads retain uridine 5′-diphospho-glucuronosyltransferase (UGT) activity. However, the contributions of individual UGT isoforms are not directly assessable in this system except through use of model inhibitors. Thus, a preparation wherein recombinant UGT (rUGT) microsomes bound to these same beads to form rUGT-beads of individual UGT isoforms would provide a novel system for measuring the contribution of individual UGT isoforms in a direct manner. To this end, the enzyme activities and kinetic parameter estimates of various rUGT isoforms in rUGT-beads were investigated, as well as the impact of fatty acids (FAs) on enzyme activity. The catalytic efficiencies () of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where is the maximum product formation rate normalized to milligram of microsomal protein (pmol/min/mg protein). Interestingly, in contrast to traditional rUGT preparations, the sequestration of UGT-inhibitory FA using bovine serum albumin did not alter the catalytic efficiency () of the rUGTs in rUGT-beads. Moreover, the increase in catalytic efficiency of rUGT-beads over rUGT microsomes was similar to increases in catalytic efficiency noted with rUGT microsomes (not bound to beads) incubated with bovine serum albumin, suggesting the beads in some way altered the potential for FAs to inhibit activity. The rUGT-bead system may serve as a useful albumin-free tool to determine kinetic constants for UGT substrates, particularly those that exhibit high binding to albumin.

中文翻译：

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确定单个尿苷 5'-二磷酸葡萄糖醛酸基转移酶 (UGT) 亚型活性的新系统：重组 UGT 珠


先前的工作表明，人肝微粒体 (HLM) 可以自发地与二氧化硅包被的磁珠 (HLM-beads) 结合，并且这些 HLM-beads 保留尿苷 5'-二磷酸-葡萄糖醛酸基转移酶 (UGT) 活性。然而，除非使用模型抑制剂，否则在该系统中无法直接评估各个 UGT 同工型的贡献。因此，其中重组UGT (rUGT)微粒体与这些相同的珠子结合以形成各个UGT同工型的rUGT-珠子的制剂将提供用于以直接方式测量各个UGT同工型的贡献的新系统。为此，研究了 rUGT 珠中各种 rUGT 亚型的酶活性和动力学参数估计，以及脂肪酸 (FA) 对酶活性的影响。与 rUGT 微粒体相比，rUGT 珠中测试的 rUGT 的催化效率 () 是 rUGT 微粒体的两倍至七倍，但 rUGT1A6 除外，其中最大产物形成率标准化为毫克微粒体蛋白 (pmol/min/mg 蛋白)。有趣的是，与传统的 rUGT 制剂相比，使用牛血清白蛋白封存 UGT 抑制性 FA 并没有改变 rUGT 珠中 rUGT 的催化效率 ()。此外，rUGT 珠子相对于 rUGT 微粒体的催化效率的增加与用牛血清白蛋白孵育的 rUGT 微粒体（未与珠子结合）所观察到的催化效率的增加相似，这表明珠子以某种方式改变了 FA 抑制的潜力。活动。 rUGT 珠系统可以作为一种有用的无白蛋白工具来确定 UGT 底物的动力学常数，特别是那些与白蛋白具有高结合力的底物。