This protocol is mainly used for the assay of α-1,2-galactosyltransferase prepared from Saccharomyces cerevisiae cells in which gma12+ gene of Schizosaccharomyces pombe, encoding an α-1,2-galactosyltransferase, is overexpressed under the control of TDH3 promoter.
Solubilized membrane fraction of yeast cells and UDP-[3H]Gal are used as an enzyme source and a donor of galactose, respectively. |
Category | Glycosyltransferases & related proteins |
Protocol Name | Enzyme assay of yeast glycosyltransferases ~ alpha-1,2-galactosyltransferase |
Authors
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Yoko-o, Takehiko
Glycoscience and Glycotechnology Research Group, Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST)
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KeyWords |
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Reagents
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UDP-Galactose (Sigma-Aldrich, St. Louis, MO) |
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UDP-[3H]Galactose (GE Healthcare, Little Chalfont, UK) |
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Dowex 1X8-400 chloride form (Sigma-Aldrich) |
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TMS buffer (20 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 0.25 M sucrose) |
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α-Methyl D-mannoside or appropriate sugar acceptors |
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Clear-sol I scintillation mixture (Nacalai Tesque Inc., Kyoto, Japan) |
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Instruments
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Bead beater (B. Braun Melsungen AG, Melsungen, Germany) |
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Ultracentrifuge (L-80 Optima, Beckman Coulter, Inc. Brea, CA) and 70.1Ti Rotor |
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Liquid scintillation counter |
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Methods |
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Preparation of solubilized microsomal proteins
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1) |
Wash 5–10 g of freshly grown cells with ice-cold water. |
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Resuspend them in 10 mL of TMS buffer. |
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Break cells by six 1 min bursts in a Bead Beater containing 0.5 mm glass beads. |
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Remove glass beads, unbroken cells and large cell debris by centrifugation (10,000 × g, 15 min, 4°C). |
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Centrifuge the resultant supernatant (100,000 × g, 60 min, 4°C). |
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Remove supernatant and resuspend pellet (microsomal fraction) in TMS buffer to a protein concentration of 20–25 μg/mL. |
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Disrupt and solubilize microsomes by the addition of Triton X-100 to a final concentration of 2% (v/v). |
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Mix gently them at 4°C for 30 min. Use of rotating mixer may be better. |
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Centrifuge the mixture (100,000 × g, 60 min, 4°C). |
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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. |
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2. |
Assay of galactosyltransferase
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1) |
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. |
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Incubate the reaction mixture at 30°C for 30–60 min. |
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Add 200 μL of ice-cold water to terminate the reaction. |
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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. |
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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). |
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Measure radioactivity of 3H by liquid scintillation counter. |
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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. |
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Notes | UDP-[3H]Galactose can not be purchased from GE Healthcare any longer. It may be available from American Radiolabeled Chemicals (ARC). |
Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-05-27 13:15:21 |
- Roy, S. K., Yoko-o, T., Ikenaga, Y., and Jigami, Y. (1998) Functional evidence for UDP-galactose transporter in Saccharomyces cerevisiae through the in vivo galactosylation and in vitro transport assay. J. Biol. Chem. 273, 2583–2590. [PMID : 9446560]
- Chappell, T. G., and Warren, G. (1989) A galactosyltransferase from the fission yeast Schizosaccharomyces pombe. J. Cell Biol. 109, 2693–2702. [PMID : 2512297]
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