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Enzyme assay of N-acetylglucosaminyltransferase V (GnT-V)
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Enzyme assay of N-acetylglucosaminyltransferase V (GnT-V)

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

Enzyme assay of N-acetylglucosaminyltransferase V (GnT-V)

Authors
Korekane, Hiroaki
Department of Disease Glycomics (Seikagaku Corporation), The Institute of Scientific and Industrial Research, Osaka University

Taniguchi, Naoyuki *
Systems Glycobiology Research Group, Chemical Biology Department, RIKEN Advanced Science Institute
*To whom correspondence should be addressed.
Reagents

100 μM PA-GnGnbi in Milli-Q water

2× GnT-V reaction buffer : 250 mM MES-NaOH buffer, 80 mM UDP-GlcNAc, 20 mM EDTA, 400 mM N-acetylglucosamine, 1% (w/v) Triton X-100, pH 6.25. Stored at −20°C until use.

Instruments

TSKgel ODS-80TM column (0.46 × 15 cm, Tosoh Corp., Tokyo, Japan)

Methods
1.

Preparation of crude enzyme extracts from tissues

1) 

 Homogenize tissues with 4 vol of 10 mM Tris-HCl (pH 7.4), 0.25 M sucrose, and proteinase inhibitors (Roche, Complete proteinase inhibitors cocktail EDTA-free).

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2) 

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

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3) 

 Collect Sup and then use for the enzyme activity assay.

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2.

Preparation of crude enzyme extracts from cultured cells

1) 

 Wash cultured cells with PBS (−) 3 times and then harvest them as cell pellets.

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2) 

 Suspend the cell pellets in 100–200 μL of ice-chilled PBS (−).

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3) 

 Sonicate the suspended cells for 5–10 min with a Bioruptor, and then use for the enzyme activity assay.

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3.

GnT-V activity assay

1) 

 Mix the following components in a small plastic tube.

2× GnT-V reaction buffer 5 μL

100 μM GnGnbi-PA 1 μL

Enzyme extracts 4 μL

Final 10 μL

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2) 

 Incubate at 37°C for 2–4 h.

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3) 

 Add 40 μL of Milli-Q water and then boil for 2 min to stop the enzyme reaction.

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4) 

 Centrifuge at 20,000 × g for 5 min to remove denatured proteins and then collect Sup.

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5) 

 Analyze 10 μL of the cleared Sup using reversed phase HPLC (Fig. 2) and then calculate the enzyme activity from the product peak area.

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Notes

For reagents:

100 μM PA-GnGnbi in Milli-Q water: the PA-acceptor oligosaccharide is obtained from hen’s egg yolk by means of purification of sialylglycopeptide (SGP) (Seko A, 1997), hydrazinolysis, re-N-acetylation, and pyridylamination (Natsuka S, 1998). Through sequential exoglycosidase digestions of the PA-SGP with sialidase and β-galactosidase, the acceptor oligosaccharide PA-GnGnbi is obtained. The PA-GnGnbi is further purified by reversed phase HPLC using a TSKgel ODS-80TM column (0.78 × 30 cm, Tosoh Corp.), which has been equilibrated with 20 mM ammonium acetate buffer pH 4.0, containing 0.25% (v/v) 1-butanol, at a flow rate of 2 mL/min, at 55°C. Elution is performed isocratically and the fluorescence of the column eluate is detected at excitation and emission wavelengths of 320 and 400 nm, respectively.

Figure & Legends

Figure & Legends

Fig. 1. Reaction catalyzed by GnT-V

 

Fig. 2. A typical chromatogram for the enzyme assay

Column; TSKgel ODS-80TM (0.46 × 15 cm)

Buffer A; 20 mM ammonium acetate pH 4.0

Buffer B; buffer A containing 1% (v/v) 1-butanol

Elution; 10% buffer B, isocratic

Flow rate; 1 mL/min

Column temp; 55°C

Fluorescence, excitation/emission=320/400 nm

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