Glycosyltransferase assays

Glycosphingolipid Glycosyltransferase Assays. A 25-33% (vol/vol) homogenate of mouse tumors or harvested cells in 0.32 M sucrose containing 0.1% 2-mer-captoethanol and 0.001 M EDTA (pH 7.0) was used as enzyme source. Membrane fractions for glycolipid gly-cosyltransferase assays were isolated at the junction of 0.32 M and 1.2 M on a discontinuous sucrose density gradient (32,43). 

Glycosyltransferase assays. Glycosyltransferase assays were carried out on sonicated fresh or frozen (-20oC) cell suspensions in 0.05 JMES. KC1 buffer pH 6.5 containing 0.OO3M MnCl2 as described previously (13). 

If the compoimd of interest has a very low reaction rate (<1% of the activity of a known standard substrate), further evaluations with concentrated enzyme are recommended. There are examples of traces of reactive substrates remaining at these low levels in inactive compoimds. Since 0.1% conversion of a reactant to product can easily be detected in radiochemical glycosyltransferase assays, confirmation that the low rate of conversion is not due to the presence of contaminants is essential. This level of contamination in modified synthetic substrates is sometimes difficult to detect even by high-field NMR spectroscopy. Therefore before the actual preparative synthesis is performed, 10% conversion of substrate to product should be verified by assay to ensure that the low rate of reaction is not due to traces of substrate in a largely inactive compound. 

TABLE I. Reacticm Mixtures and Incubation Conditions for Glycosyltransferase Assays"... 

The present review covers a description of methodology and properties of UDP-glucuronyltransferase and of related UDP-glycosyltransferase activities (assayed with bilirubin as the acceptor substrate) and attempts to delineate applications to human disease. Studies with other hydro-phobic acceptor substances will be discussed as far as relevant to the subject matter. 

Obviously, extrapolation procedures are impractical for routine determination of enzyme activities. When substrate saturation-curves conform to rectangular hyperbolas, reasonable concentrations of substrates should equal 10 to 20 times the respective Km values. As outlined above, application of this rule to assays of bilirubin UDP-glycosyltransferase activities is hampered by substrate inhibition and by occasional deviation from Michaelis-Menten kinetics. The best alternative in such cases may be to choose the concentrations at optimal enzyme activity. However, great care should be exercised in interpreting the results. When a bio-... 

Procedures for Determination of Conjugated Bilirubin Applicable to the Assay of Bilirubin UDP-Glycosyltransferase... 

N4TG1 cells were exposed to drugs for 8 hrs. and the homogenates assayed for glycosyltransferase activity as described in the text... 

Persson M, Palcic MM (2008) A high-throughput pH indiciator assay for screening glycosyltransferase saturation mutagenesis libraries. Anal Biochem 378 1-7... 

Schwartz B, Markwalder JA, Seitz SP, Wang Y, Stein RL. A kinetic characterization of the glycosyltransferase activity of Eschericia coli PBP lb and development of a continuous fluorescence assay. Biochemistry 2002 41 12552-12561. 

After the glycosyltransferase of interest has been pmchased or isolated, it is desirable to verify the activity of the enzyme with a standard substrate and the compound of interest under the conditions of the preparative synthesis. Methods for assaying glycosyltransferase activity are summarized in Table 1, and a suitable method should be selected based on the characteristics of the substrate and the required sensitivity of the assay. Kinetic studies to obtain Km and Kiax values for the substrate and optimization of reaction conditions, including buffer and pH, should also be carried out. 

The activity of glycosyltransferases can also be monitored throughout the reaction comse by removing a small aliquot from the reaction, followed by dilution and assay of activity by one of the standard assays with defined substrates in Table 1. It is desirable to monitor the pH of the reaction closely, especially when the concentration of the substrate is relatively high compared to molarity of the buffer. The pH of glycosyltransferase reactions drops as reactions proceed as a result of the production of phosphoric acid by alkaline phosphatase. Acidic pH values can inactivate glycosyltransferases and substrates, and decrease the efficiency of alkaline phosphatase. 

---