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Recombinant Mouse ST3GAL5/GM3 Synthase Protein, CF

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Summary
Reactivity MuSpecies Glossary
Applications Enzyme Activity
Format
Carrier-Free

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Recombinant Mouse ST3GAL5/GM3 Synthase Protein, CF Summary

Details of Functionality
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.
Source
Chinese Hamster Ovary cell line, CHO-derived mouse ST3GAL5/GM3 Synthase protein
Thr87-His414, with an N-terminal 6-His tag
Accession #
N-terminal Sequence
His
Protein/Peptide Type
Recombinant Enzymes
Gene
St3gal5
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Endotoxin Note
<0.1 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

Dilutions
  • Enzyme Activity
Theoretical MW
38 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
SDS-PAGE
45-60 kDa, reducing conditions

Packaging, Storage & Formulations

Storage
Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
Buffer
Supplied as a 0.2 μm filtered solution in Tris and NaCl.
Purity
>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • 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 # EA002)
  • 96-well Clear Plate (Catalog # DY990)
  • 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

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Mouse ST3GAL5/GM3 Synthase Protein, CF

  • 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

Background

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).
  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.

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Bioinformatics

Gene Symbol St3gal5
Uniprot