Purity
>95%, by SDS-PAGE under reducing conditions and visualized by silver stain
Endotoxin Level
<1.0 EU per 1 μg of the protein by the LAL method.
Activity
Measured by its ability to hydrolyze the substrate 4-Nitrocatechol Sulfate (PNCS). The specific activity is >0.4 pmol/min/µg, as measured under the described conditions. See Activity Assay Protocol on .
Source
Spodoptera frugiperda, Sf 21 (baculovirus)-derived Arg23-Leu502, with a C-terminal 10-His tag
Accession #
N-terminal Sequence
AnalysisArg23
Predicted Molecular Mass
56 kDa
SDS-PAGE
60 kDa, reducing conditions
2969-SU |
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Formulation Supplied as a 0.2 μm filtered solution in Tris and NaCl. | ||
Shipping The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below. | ||
Stability & Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
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Assay Procedure
Materials
Assay Buffer: 50 mM MES, pH 5.5
Recombinant Mouse Sulfamidase/SGSH (mSGSH) (Catalog # 2969-SU)
Substrate: 4-Nitrocatechol Sulfate (PNCS) (Sigma, Catalog # N-7251), 100 mM stock in deionized water
Sodium Hydroxide (NaOH) (Sigma, Catalog # 221465), 2 M stock in deionized water
96-well Clear Plate (Costar, Catalog # 92592)
Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
Dilute rmSGSH to 40 µg/mL in Assay Buffer.
Dilute Substrate to 20 mM in Assay Buffer.
Mix 150 µL of 40 µg/mL of rmSGSH and 150 µL of 20 mM of Substrate. Include a Substrate Blank with 150 µL of Assay Buffer and 150 µL of Substrate.
Incubate at 37 °C for 72 hours.
Stop reaction by adding 300 µL of 0.2 M NaOH to the reaction vials.
Load in a plate 200 µL in duplicate from each reaction vial.
Read at 510 nm (absorbance) in endpoint mode.
Calculate specific activity:
Specific Activity (pmol/min/µg) = | Adjusted Abs* (OD) x Conversion Factor** (pmol/OD) |
| Incubation time (min) x amount of enzyme (µg) |
*Adjusted for Substrate Blank
**Derived using calibration standard P-Nitrocatechol (PNC) (Sigma, Catalog # N15553).
Per Well:
rmSGSH: 2 µg
Substrate: 5 mM
Background: Sulfamidase/SGSH
Also known as N-sulfoglucosamine sulfohydrolase and heparan N-sulfatase, Sulfamidase/SGSH is an important member of the sulfatase family involved in the degradation of heparan sulfate (1). The SGSH deficiency results in mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome), an autosomal recessive lysosomal storage disease characterized by neurological dysfunction but relatively mild somatic manifestations (2). The deduced amino acid sequence of mouse SGSH consists of a signal peptide (residues 1-22) and a mature chain (residues 23-502) (1). Recombinant Mouse Sulfamidase/SGSH corresponds to the mature chain and has sulfatase activity as described in the Activity section.
References:
Diez-Roux, G. and Ballabio, A. (2005) Annu. Rev. Genomics Hum. Genet. 6:355.
Gliddon, B.L. et al. (2004) Mol. Genet. Metab. 83:239.
Entrez Gene IDs:
6448 (Human); 27029 (Mouse)
Alternate Names:
heparan sulfate sulfatase; HSS; HSSN-sulphoglucosamine sulphohydrolase; MPS3A; MPS3Asulphamidase; N-sulfoglucosamine sulfohydrolase; SFMD; SFMDEC 3.10.1.1; SGSH; Sulfamidase; Sulfoglucosamine sulfamidase; Sulphamidase
