The non-radioactive phosphatase-coupled glycosyltransferase assay described here offers a new method for measuring glycosyltransferase activity. This assay couples specific phosphatases with glycosyltransferase reactions to quantitatively release inorganic phosphate from the leaving nucleoside phosphates produced during the glycosyltransferase reactions. The released phosphate group is then detected using colorimetric malachite-based reagents. Because the amount of phosphate released is directly proportional to the sugar molecule transferred in a glycosyltransferase reaction, this method can be used to obtain accurate kinetic parameters of the glycosyltransferase. The assay can be performed in multi-well plates and quantitated by a plate reader, making it amenable to high-throughput screening. It has been successfully applied to all glycosyltransferases available to us, including glucosyltransferases, N-acetylglucosaminyltransferases, galactosyltransferases, N-acetylgalactosyltransferases, fucosyltransferases, and sialyltransferases. The method is demonstrated using Clostridium difficile toxin B (a protein O-glucosyltransferase), human KTELC1 (a glucosyltransferase homolog to Rumi from Drosophila) and human sialyltransferase ST6GAL1.
STEP 1: In a 96-well plate, prepare a reaction mix containing the donor, acceptor substrate, and coupling phosphatase in buffer.
STEP 2: Initiate the reaction by adding a specific glycosyltransferase.
STEP 3: Stop the reaction and develop the color using Malachite Green Phosphate detection reagents. Read the absorbance at 620 nm with a plate reader.
Reaction Buffer: 25 mM Tris, 150 mM MgCl2, 5mM MnCl2 at pH 7.5.
|Figure 1. CMP (green), GDP (purple) and UDP (blue), were treated with CD73 (A), CD39L3 (B) and TNAP (C, D). Reactions with phosphate content higher than 4 nmol were diluted before measurement and absorbances were obtained by multiplying the observed O.D.s by the dilution factors. Dashed red lines represent phosphate standard curves.|
|Figure 2. All reactions were carried out for 20 minutes at 37 °C (A) Specific activity (SA) versus UDP-Glc. (B) Activity versus enzyme dose in the presence of 1 mM UDP-Glc.|
|Figure 3. Each reaction was coupled to 0.1 μg of CD39L3. All reactions were carried out for 2 hours at 37 °C (A) Specific activity (SA) versus UDP-Glc. (B) Activity versus enzyme dose in the presence of 0.2 mM UDP-Glc.|
|Figure 4. Each reaction was coupled to 0.1 μg of CD73. (A) Specific activity (SA) versus donor substrate CMP-NeuAc in the presence of 1 mM acceptor LN. (B) Specific activity versus acceptor substrate LN in the presence of 0.2 mM CMP-NeuAc. (C) Activity versus enzyme dose in the presence of 2 mM CMP-NeuAc and 8 mM LN.|
For research use only. Not for use in diagnostic procedures.