UGT Activity Assay / Ligand Screening Kit (Fluorometric)

Uridine diphospho-glucuronosyltransferases (UGTs, EC 2.4.1.17) are a superfamily of microsomal membrane-bound glycosyltransferase enzymes. UGTs are responsible for the vast majority of Phase II biotransformation (conjugation) reactions, in which a hydrophilic moiety is attached to small molecule drugs and other xenobiotics to facilitate their rapid excretion from the body. UGTs are predominantly expressed in the liver, intestine and kidneys, where they catalyze the addition of a glucuronic acid moiety from the nucleotide sugar uridine-5’-diphospho-α-D-glucuronic acid (UDPGA) to nucleophilic hydroxyl, sulfhydryl or amino groups of pharmacologically active drugs, Phase I drug metabolites and certain endogenous substrates such as steroid hormones, bile acids and bilirubin. Isozymes of the UGT family have extremely broad, often overlapping substrate specificities and tend to exhibit modest affinity for substrates but high catalytic turnover rates. UGTs also contribute to ‘first-pass’ presystemic drug metabolism, which dramatically reduces the oral bioavailability of a number of drugs. BioVision’s UGT Activity Assay / Ligand Screening Kit enables rapid measurement of native or recombinant UGT activity in biological samples such as liver microsomes and can also be used to assess the effect of drugs and other novel compounds on UGT activity. The assay utilizes a highly fluorescent UGT substrate with a large Stokes shift (Ex/Em = 415/502 nm) that allows determination of UGT activity by tracking the drop in fluorescence emission as the substrate is converted into a non-fluorescent glucuronide. The multi-isozyme substrate is glucuronidated by virtually all of the pharmacologically-relevant mammalian UGT1A and UGT2B enzymes. UGT specific activity is calculated by comparing the fluorescence loss versus a control reaction performed in the absence of the required cofactor UDPGA. The kit includes the pore-forming peptide antibiotic Alamethicin, which allows the UGT Substrate and UDPGA to rapidly diffuse across lipid membranes to access the UGT active site located in the lumen of microsomes. For verification of modulation of UGT activity by test ligands, diclofenac, a competitive inhibitor of most human and rodent UGT isozymes, is also included. The assay is highly sensitive, simple to perform and high-throughput adaptable. This assay can detect less than 0.1 mU UGT activity in biological samples. The kit contains a complete set of reagents sufficient for performing 100 reactions at a 100 μl reaction volume