Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute 1 mM Phosphate Standard by adding 40 µL of the 1 mM Phosphate Standard to 360 µL of Assay Buffer for a 100 µM stock. This is the first point of the standard curve.
Continue 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 5.0 nmol per well.
Load 50 µL of each dilution of the standard curve into a plate. Include a curve blank containing 50 μL of Assay Buffer.
Prepare a Reaction Mixture composed of 1 mM UDP-GlcA, 1 mM UDP-GlcNAc, 8 mg/mL Heparan Sulfate, and 4 µg/mL Coupling Phosphatase 1 in Assay Buffer.
Dilute rhEXT1 to 4 µg/mL in Assay Buffer.
Load 25 µL of the 4 µg/mL rhEXT1 into the plate. Include a Substrate Blank containing 25 µL of Assay Buffer.
Start the reaction by adding 25 µL of Reaction Mixture to the wells, excluding the standard curve and curve blank.
Cover the plate with a plate sealer and incubate at 37 °C for 20 minutes.
Add 30 µL of the Malachite Green Reagent A to all wells. Mix briefly.
Add 100 µL of deionized water to all wells. Mix briefly.
Add 30 µL of the Malachite Green Reagent B to all wells. Mix and incubate for 20 minutes at room temperature.
Read plate at 620 nm (absorbance) in endpoint mode.
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.
rhEXT1: 0.1 µg
UDP-GlcA: 0.5 mM
UDP-GlcNAc: 0.5 mM
Heparan Sulfate: 200 µg
Coupling Phosphatase 1: 0.1 µg
Background: Exostosin 1
The exostosin (EXT) family of glycosyltransferases are involved in heparan sulfate (HS) and heparin biosynthesis (1). Mutations of these enzymes are the causes of hereditary multiple exostoses, which are characterized by formation of numerous cartilage-capped, benign bone tumors (osteocartilaginous exostoses or osteochondromas) that are often accompanied by skeletal deformities and short stature (2). In a small percentage of cases exostoses have exhibited malignant transformation resulting in an osteosarcoma or chondrosarcoma (3). Five members of this family have been cloned to date: EXT1, EXT2, EXTL1, EXTL2, and EXTL3. EXT1 and EXT2 are bifunctional enzymes with both beta -1,4-glucuronyltransferase and alpha -1,4-N-acetylglucosaminyltransferase activities that add alternating beta -1,4-GlcA and alpha -1,4-GlcNAc residues on the non-reducing end of HS chain (4). EXT1 is an active enzyme, but the hetero-dimer of EXT1 and EXT2 is more stable and has higher activity (1). The enzyme activity of recombinant human EXT1 is measured using a phosphatase-coupled assay (5).
Busse, M. et al. (2007) J. Biol. Chem. 282:32802.
Wuyts, W. et al. (1998) Am. J. Hum. Genet. 62:346.
Hecht J.T. et al. (1997) Am. J. Hum. Genet. 60:80.
Kobayashi S. et al. (2000) Biochem. Biophys. Res. Commun. 268:860.
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