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Enzyme assay of yeast glycosyltransferases ~ alpha-1,6-mannosyltransferase (Och1p)
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Enzyme assay of yeast glycosyltransferases ~ alpha-1,6-mannosyltransferase (Och1p)

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Category
Glycosyltransferases & related proteins
Protocol Name

Enzyme assay of yeast glycosyltransferases ~ alpha-1,6-mannosyltransferase (Och1p)

Authors
Nakayama, Ken-ichi
Biomass Treatment Group, Research Institute for Sustainable Chemistry, National Institute of Advanced Industrial Science and Technology (AIST)
KeyWords
Reagents

Yeast cells (OCH1 wild type)

Yeast Extract (BD, Franklin Lakes, NJ)

Bacto Peptone (BD)

Glucose (Wako Pure Chemical Industries Ltd., Osaka, Japan)

50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 1 mM PMSF, 5% glycerol and 2 μg/mL each of proteinase inhibitor (antipain, chymostatin, leupeptin and pepstatin A)

Glass beads (0.45–0.50 ø mm)

GDP-mannose (Sigma-Aldrich, St. Louis, MO)

Man8GlcNAc2-PA (Takara Bio Inc., Otsu, Japan)

Asahipak NH2P-50 (0.46 × 25 cm) (Showa Denko, K.K., Tokyo, Japan)

200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 3 : 7

200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 7 : 3

Instruments

Incubator

Homogenizer (B.Braun or Vortex mixer)

Centrifuge

Ultra centrifuge

HPLC system

Fluorescent detector

Methods
1.

Preparation of yeast membranes and alpha-1,6-mannosyltransferase assay

1) 

 Yeast cells are grown in 250 mL of YPAD medium at 30˚C for overnight.

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2) 

 Collect cells by centrifugation at 3000 × g for 5 min and wash with 1% KCl.

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3) 

 Resuspend cells in 5 mL of 50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 1 mM PMSF, 5% glycerol and 2 mg/mL each of proteinase inhibitor.

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4) 

 Add glass beads (0.45–0.50 ø mm) to half of cell suspension volume and homogenize with B. Braun homogenizer for 1 min × 3 times at 4˚C.

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5) 

 Homogenates are filtrated by G1 glass filter and centrifuged at 10,000 × g for 20 min.

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6) 

 Supernatant is further centrifuged at 100,000 × g for 1 h.

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7) 

 Collect pellet as HSP (high speed pellet).

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8) 

 HSP are resuspended in a buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 1 mM PMSF, 5% glycerol, and proteinase inhibitor mixture.

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9) 

 A 200 μg protein of HSP is incubated in 50 mL of 50 mM Tris-HCl (pH 7.5), 10 mM MnCl2, 0.6% Triton X-100, 0.5 mM 1-deoxy-mannojirimycin, 20 pmol Man8GlcNAc2-PA, 1 mM GDP-mannose at 30˚C for 30 min.

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10) 

 Boil for 5 min and reaction mixture is centrifuged at 15,000 rpm for 5 min.

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11) 

 Supernatant of reaction mixture is analyzed by HPLC.

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2.

HPLC analysis

1) 

 Equilibrium buffer is a mixture of 95 % of solvent A (200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 3 : 7) and 5% of Solvent B (200 mM acetic acid / triethylamine pH 7.3 : acetonitrile = 7 : 3).

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2) 

 Elution conditions are as follows:

Column: Asahipak NH2P-50 (0.46 × 25 cm)

Column temperature: 25˚C

Flow rate: 1.0 mL/min

Elution condition: linear gradient of Solvent B from 5% to 45% between 0–50 min

Fluorescent detector: Excitation = 310 nm, Emission = 380 nm

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Figure & Legends

Figure & Legends

 

 

Fig. 1.  Analysis of Och1p activity. A; Man8GlcNAc2-PA (acceptor).  B; Man9GlcNAc2-PA (Och1p reaction product).

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