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Enzyme assay of α1,3/4-fucosyltransferase
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Enzyme assay of α1,3/4-fucosyltransferase

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Introduction Protocol References Credit lines
Category
Glycosyltransferases & related proteins
Protocol Name

Enzyme assay of α1,3/4-fucosyltransferase

Authors
 Kudo, Takashi
Department of Anatomy and Embryology, University of Tsukuba
KeyWords
Reagents
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Sodium cacodylate

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ATP

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MnCl2

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L-fucose

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GDP-fucose

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Pyridylaminated (PA)-sugar : (sialyl α 2,3-)LNnT(lacto-N-neotetraose; Galβ1-4GlcNAcβ1-3Galβ1-4Glc)-PA for α1,3-FUT assay, (sialyl α 2,3-)LNT(lacto-N-tetraose; Galβ1-3GlcNAcβ1-3Galβ1-4Glc)-PA for α1,4-FUT assay) (Takara Bio Inc., Otsu, Japan)

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Cell extracts as enzyme sources (from FUT transfected cell lines (ex. COS-1, HEK293T)

Instruments
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Ultrasonic bath sonicator (Cosmo Bio Co., Ltd., Tokyo, Japan)
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Water bath
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Ultracentrifuge
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HPLC
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TSK-gel ODS-80TS column (4.6 mm × 250 mm, Tosoh Corp., Tokyo, Japan)
Methods
1.

Preparation of crude enzyme extracts from transfectant cells

1) 

 Solubilize fucosyltransfease (over-) expressing cells into 20 mM HEPES (pH 7.4), 0.1% Triton-X100 by sonicating in an ultrasonic bath sonicator for 10 min.

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2) 

 Centrifuge at 600 × g for 5 min at 4°C.

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3) 

 Collect supernatant and use as the enzyme source for fucosyltransferase activity.

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2.

α1,3/4-fucosyltransferase assay

1) 

 Mix the following components in a small plastic tube.

1 M cacodylate buffer (pH 6.8) 0.5 μL (Final conc. 50 mM)

250 mM MnCl2 1 μL (Final conc. 25 mM)

100 mM ATP 0.5 μL (Final conc. 5 mM)

75 μM GDP-fucose 1 μL (Final conc. 7.5 μM)

100 mM L-fucose 1 μL (Final conc. 10 mM)

0.5 mM PA-sugar 0.5 μL (Final conc. 0.025 mM)

solubilized extracts 5.5 μL

total 　　　 10 μL

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2) 

 Incubate at 37˚C for 2 h.

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3) 

 Centrifuge at 20,000 × g for 5 min at 4°C.

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4) 

 Analyze 10 μL of the supernatant using high pressure liquid chromatography on a TSK-gel ODS-80TS column.

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5) 

 Elute the reaction products with 20 mM ammonium acetate buffer (pH 4.0) at the flow rate of 1.0 mL/min at 35°C.

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6) 

 Monitor with a fluorescence spectrophotometer (FP-920, JASCO Corporation, Tokyo, Japan) and calculate the fucosyltransferase activity from the product peak areas.

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Notes

α2,3-sialyl-LNT-PA and α2,3-sialyl-LNnT-PA were synthesized by sialylation of LNT-PA and LNnT-PA using a recombinant α2,3-saialyltransferase (ST3Gal3, ST3Gal4).

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