Enzyme assay of glycolipid glycosyltransferases ~GD3 synthase (α2,8-sialyltransferase)

Authors：

Category

Glycosyltransferases & related proteins

Protocol Name

Authors

Furukawa, Koichi *
Department of Biochemistry II, Nagoya University Graduate School of Medicine

Furukawa, Keiko
Deparment of Biomedical Sciences, College of Life and Health Sciences, Chubu University

*To whom correspondence should be addressed.

KeyWords

Membrane preparation Sugar nucleotides Cations Substrates Glycolipids

Reagents

●	Sodium cacodylate (Wako Pure Chemical Industries, Ltd., Osaka, Japan)
●	CMP-NeuAc (Sigma-Aldrich, St. Louis, MO)
●	GM3 as an acceptor (Sigma-Aldrich, St. Louis, MO)
●	[¹⁴C]-CMP-NeuAc (NEN/PerkinElmer, Waltham, MA)
●	Extracts as an enzyme source
●	Triton CF54 (Sigma-Aldrich)

Instruments

●	N₂ cavitation apparatus
●	Water bath
●	N₂ evaporator
●	TLC plate
●	Imaging analyzer
●	Ultracentrifuge and swing type bukette
●	SepPak C18 cartridge (Waters Corp., Milford, MA)

Methods

Preparation of membrane fraction

1)	N₂ cavitation of cell pellets at 400 psi on ice for 30 min.	Comment 1

2)	Centrifuge at 1,000 rpm for 10 min at 4°C.	Comment 0

3)	Ultracentrifuge the supernatant at 34 K (Beckman, SW 55Ti) for 1 h at 4°C.	Comment 0

Resuspend the pellets in 0.1 M cacodylate buffer, pH 6.0.

Comment 0

Enzyme assay

1)	Evaporate the following items in a glass tube. CMP-NeuNc (500 μM) GM3 (325 μM) [¹⁴C]-CMP-NeuNc (9.2Å-103 dpm)	Comment 0

2)	Dissolve in 0.05 M cacodylate buffer (adjust to make final volume 50 μL).	Comment 0

3)	Add 5 mM MnCl₂, 0.3% Triton CF54 in cacodylate buffer.	Comment 0

4)	Sonicate for 10 sec.	Comment 0

5)	Add membrane proteins (200 μg).	Comment 0

6)	Incubate at 37°C for 2-3 h with shaking.	Comment 0

7)	Add DW 1 mL to stop the reaction.	Comment 0

8)	Separate the products by SepPak C18 column.	Comment 0

9)	Dry with N₂ stream.	Comment 0

10)	Count radioactivity of 1/5 of the products by scintillation counter.	Comment 0

11)

Analyze 4/5 of the products in TLC and autoradiography.

Comment 1

Notes

This protocol was reported in the previous article (Furukawa K. et al. 2009).

Enzyme reaction of GD3 synthase was shown in Fig. 1.

Figure & Legends

Fig. 1. Enzyme reaction of GD3 synthase (ST8SiaI)

A precursor structure GM3, sugar nucleotide CMP-NeuAc (donor substrate) and enzyme source are essential. Detergent (Triton CF54) and MnCl₂ increase enzyme activity.

This figure was originally published in Glycoconj J. 26(8), 2009, 987-98, "Glycoconjugate glycosyltransferases", Furukawa K. et al. Fig.1; With kind permission of Springer Science and Business Media.

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Date of registration：2013-12-24 16:48:29