• Purity

  >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <0.1 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to transfer Neu5Ac from CMP-Neu5Ac to alpha -lactose. The specific activity is >5 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Chinese Hamster Ovary cell line, CHO-derived Thr87-His414, with an N-terminal 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  His

• Predicted Molecular Mass

  38 kDa

• SDS-PAGE

  45-60 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: 25 mM MES, pH 7.0

• Recombinant Mouse ST3GAL5/GM3 Synthase (rmST3GAL5) (Catalog # 8454-GT)

• Donor Substrate: CMP-Sialic Acid (CMP-Neu5Ac) (Sigma, Catalog # C8271), 10 mM stock in deionized water

• Acceptor Substrate: alpha -Lactose (Sigma, Catalog # L2643), 300 mM stock in deionized water

• Sialyltransferase Activity Kit (Catalog # )

• 96-well Clear Plate (Catalog # )

• Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent

1. Dilute 1 mM Phosphate Standard provided by the Sialyltransferase Activity Kit by adding 40 μL of the 1 mM Phosphate Standard to 360 μL of Assay Buffer for a 100 μM stock.

2. Prepare standard curve by performing six one-half serial dilutions of the 100 μM Phosphate stock in Assay Buffer. The standard curve has a range of 0.078 to 5 nmol per well.

3. Prepare reaction mixture containing 0.4 mM CMP-Sialic Acid, 80 mM alpha -lactose, and 2 μg/mL Coupling Phosphatase 2.

4. Dilute rmST3GAL5 to 40 μg/mL in Assay Buffer.

5. Load 50 μL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.

6. Load 25 μL of the 40 μg/mL rhST6GAL1 into the plate. Include a Control containing 25 μL of Assay Buffer.

7. Add 25 μL of reaction mixture to the wells, excluding the standard curve.

8. Cover the plate with parafilm or a plate sealer and incubate at room temperature for 3 hours.

9. Add 30 μL of the Malachite Green Reagent A to all wells. Mix briefly.

10. Add 100 μL of deionized water to all wells.

11. Add 30 μL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room at room temperature.

12. Read plate at 620 nm (absorbance) in endpoint mode.

13. Calculate specific activity:

    Specific Activity (pmol/min/μg) =	Phosphate released* (nmol) x (1000 pmol/nmol)
    	Incubation time (min) x amount of enzyme (μg)

         *Derived from the phosphate standard curve using linear or 4-parameter fitting and adjusted for Control.

Per Reaction:

• rmST3GAL5: 1.0 μg

• Coupling Phosphatase 2: 0.05 μg

• CMP-Sialic Acid: 0.2 mM

• alpha -Lactose: 40 mM

Background: ST3GAL5/GM3 Synthase

Sialyltransferases add sialic acid to glycoproteins or glycosphingolipids and play important roles in many biological processes including immune recognition, pathogen infection, and cell adhesion (1). ST3GAL5 is a type II membrane protein localized in the trans-Golgi network that can transfer sialic acid to lactosylceramide (LacCer) to form GM3 (NeuAc alpha 2-3Gal beta 1-Glc beta 1-Ceramide), the main ganglioside in human plasma (2). GM3 is known to participate in differentiation, proliferation and maintenance of fibroblast morphology (3). It also plays role in signal transduction, and integrin-mediated cell adhesion. Mutation of ST3GAL5 has been associated with Amish infantile epilepsy syndrome (4) and type 2 diabetes (5). Despite the ubiquitous distribution of ganglioside GM3 in human tissues, the expression of ST3GAL5 is found in a tissue-specific manner, with predominant expression in brain, skeletal muscle, and testis, and very low expression in liver (2). The enzymatic activity of recombinant mouse ST3GAL5 was determined using a phosphatase-coupled glycosyltransferase assay (6).

• References:

1. Varki, A. (1999) Glycobiology 2:25.

2. Ishii, A. et al (1998) J. Biol. Chem. 273:31652.

3. Uemura, S. et al (2009) Mol. Biol. Cell 20:3088.

4. Simpson, M. A. et al. (2004) Nat. Genet. 36:1225.

5. Tagami, S. et al. (2002) J. Biol. Chem. 277:3085.

6. Wu, Z.L. et al. (2011) Glycobiology 21:727.

• Long Name:

  ST3 beta-Galactoside alpha-2,3-Sialyltransferase 5

• Entrez Gene IDs:

  8869 (Human); 20454 (Mouse); 83505 (Rat)

• Alternate Names:

  3S-T; alpha 2,3-sialyltransferase V; CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase; EC 2.4.99.9; Ganglioside GM3 Synthase; GM3 synthase; lactosylceramide alpha-2,3-sialyltransferase; SATI; sialyltransferase 9 (CMP-NeuAc:lactosylceramide alpha-2,3-sialyltransferase;GM3 synthase); Sialyltransferase 9; Siat9; SIATGM3S; ST3 beta-galactoside alpha-2,3-sialyltransferase 5; ST3Gal V; ST3GAL5; ST3GALV; ST3GalVSIAT9SATI