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

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Category
Glycosyltransferases & related proteins
Protocol Name

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

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

Keiko Furukawa
Deparment of Biomedical Sciences, College of Life and Health Sciences, Chubu University
*To whom correspondence should be addressed.
Keywords

Glycolipids
Substrates
Cations
Membrane preparation
Sugar nucleotides

Reagents
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Sodium cacodylate (Wako pure chemical)

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CMP-NeuAc (Sigma)

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GM3 as an acceptor (Sigma)

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[14C]-CMP-NeuAc (NEN)

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Extracts as an enzyme source

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Triton CF54 (Sigma)

Instruments
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N2 cavitation apparatus
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Water bath
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N2 evaporator
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TLC plate
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Imaging analyzer
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Ultracentrifuge and swing type bukette
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SepPak C18 cartridge (Water)
Methods
1.

Preparation of membrane fraction

1) 

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

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2) 

 Centrifuge at 1,000 rpm for 10 min at 4°C.

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3) 

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

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4) 

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

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2.

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)
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2) 

 Dissolve in 0.05 M cacodylate buffer (adjust to make final volume 50 μl).

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3) 

 Add 5 mM MnCl2, 0.3% Triton CF54 in cacodylate buffer.

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4) 

 Sonicate for 10 sec.

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5) 

 Add membrane proteins (200 μg).

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6) 

 Incubate at 37°C for 2–3 h with shaking.

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7) 

 Add DW 1 ml to stop the reaction.

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8) 

 Separate the products by SepPak C18 column.

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9) 

 Dry with N2 stream.

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10) 

 Count radioactivity of 1/5 of the products by scintillation counter.

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11) 

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

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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

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：2012-11-08 16:51:27
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