A novel system to determine activity of individual uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms: Recombinant UGT-beads

• Published in: The Journal of biological chemistry 2024-05, Vol.300 (5), p.107278-107278, Article 107278
• Main Authors: Wang, Ting, Taub, Mitchell E., Chan, Tom S.
• Format: Article
• Language: English
• Subjects: BSA; enzyme kinetics; fatty acids; human glucuronosyltransferase; immobilization; magnetizable beads; Methods and Resources; recombinant UGT; rUGT-beads
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/j.jbc.2024.107278

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 (Vmax/Km) of the tested rUGTs were twofold to sevenfold higher in rUGT-beads compared with rUGT microsomes, except for rUGT1A6, where Vmax 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 (Vmax/Km) 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.