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Enzyme assay of glycolipid glycosyltransferases ~Gb3/CD77 synthase
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Enzyme assay of glycolipid glycosyltransferases ~Gb3/CD77 synthase

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

Enzyme assay of glycolipid glycosyltransferases ~Gb3/CD77 synthase

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
Reagents
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Sodium cacodylate (Wako Pure Chemical Industries Ltd., Osaka, Japan)

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UDP-Gal (Sigma-Aldrich, St. Louis, MO)

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Lactosylceramide as an acceptor (Sigma-Aldrich, St. Louis, MO)

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[14C]-UDP-Gal (NEN/Perkin Elmer, Waltham, MA)

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Extracts as an enzyme source

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Triton X-100 (Sigma-Aldrich, St. Louis, MO)

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Galactonolactone

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Phosphatidylglycerol (Sigma-Aldrich, St. Louis, MO)

Instruments
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N2 cavitation apparatus
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Water bath
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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 (Waters Corp., Milford, MA)
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 0.05 M cacodylate HCl, pH 6.0.

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2.

Enzyme assay

1) 

 Evaporate the following items in a glass tube.

  • UDP-Gal (200 mM)
  • LacCer (0.4 mM)
  • [14C]-UDP-Gal (2.5Å-105 dpm)
  • galactonolactone (5 mM)
  • phosphatidylglycerol (2.9 mM)
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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 10 mM MnCl2, 0.3% Triton X-100 in cacodylate buffer.

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4) 

 Sonicate for 10 sec.

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5) 

 Add membrane proteins (50 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.

 Comment 1
Notes

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

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Date of registration：2013-12-24 16:42:56
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