Enzyme assay of yeast glycosyltransferases ~ alpha-1,2-galactosyltransferase

 Wash 5–10 g of freshly grown cells with ice-cold water.

 Resuspend them in 10 mL of TMS buffer.

 Break cells by six 1 min bursts in a Bead Beater containing 0.5 mm glass beads.

 Remove glass beads, unbroken cells and large cell debris by centrifugation (10,000 × g, 15 min, 4°C).

 Centrifuge the resultant supernatant (100,000 × g, 60 min, 4°C).

 Remove supernatant and resuspend pellet (microsomal fraction) in TMS buffer to a protein concentration of 20–25 μg/mL.

 Disrupt and solubilize microsomes by the addition of Triton X-100 to a final concentration of 2% (v/v).

 Mix gently them at 4°C for 30 min. Use of rotating mixer may be better.

 Centrifuge the mixture (100,000 × g, 60 min, 4°C).

 Recover supernatant. It is used as the source of solubilized enzyme. This enzyme is stable at 4°C without any protease inhibitor for several days.

 Make 50 μL of reaction mixture containing the following reagents:-

• 0.1 M HEPES-NaOH (pH 7.0)

• 1 mM MnCl₂

• 25 nmol of UDP-[³H]Gal (specific activity ~1.48 GBq/mmol)

• 5 μmol of given sugar acceptor

• Solubilized enzyme fraction

To save radioisotope, UDP-[³H]Gal may be diluted to 37–74 MBq/mmol of specific activity by unlabeled UDP-Gal. The reaction mixture contains 0.1–0.5% (v/v) Triton X-100, depending on the detergent concentration in the enzyme fraction.

 Incubate the reaction mixture at 30°C for 30–60 min.

 Add 200 μL of ice-cold water to terminate the reaction.

 Load the mixture onto a 1 mL of Dowex-1 (chloride form, 200–400 dry mesh) anion exchange column packed in a 2- or 3-mL syringe.

 Wash column twice with 1 mL water and mix the combined eluents (total 2 mL) with 2.5 volumes of scintillation mixture (Clear sol I).

 Measure radioactivity of ³H by liquid scintillation counter.

 The Dowex columns can be regenerated by sequential washing with 2.5 mL of 5 M NaCl, 2.5 mL of 0.1 M HCl and then 8 mL of water.