• Purity

  >70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie? Blue stain at 5 μg per lane

• Endotoxin Level

  <1.0 EU per 1 μg of the protein by the LAL method.  

• Activity

  Measured by its ability to form CBZ-Gln-Gly-Hydroxamate from CBZ-Gln-Gly and Hydroxylamine. The specific activity is >100 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Met2-Ala817, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Inconclusive. Gly6, Ser7, Thr491, Ala571 identified. Poly His detected by Western blot.

• Predicted Molecular Mass

  89 kDa

• SDS-PAGE

  75-88 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: deionized water

• Recombinant Human Transglutaminase 1/TGM1 (rhTGM1) (Catalog # 7765-TG)

• Substrate: Z-Gln-Gly (Sigma, Catalog # C6154), 500 mM, pH 9.0 in deionized water

• Hydroxylamine Hydrochloride (Sigma, Catalog # 159417), 1 M, pH 6.0 in deionized water

• Stop solution: 0.37 M FeCl₃ (Sigma, Catalog # 236489), 0.67 M HCl, 0.2 M Trichloroacetic acid

• 96-well Clear Plate (Costar, Catalog # 92592)

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

1. Prepare Substrate Mixture containing 62.5 mM Substrate, 250 mM MES pH 6.0, 12.5 mM DTT, 12.5 mM CaCl₂, and 125 mM Hydroxylamine Hydrochloride.

2. Dilute rhTGM1 to 0.2 mg/mL in Assay Buffer.

3. Mix 30 μL dilute rhTGM1 with 120 μL Substrate Mixture. Include an Enzyme Control containing 30 μL Assay Buffer with 120 μL Substrate Mixture.

4. Incubate at 37 °C for 2 hours.

5. Stop each reaction with 600 μL of Stop Solution. Mix well.

6. Centrifuge at top speed for 2 minutes in a microcentrifuge.

7. Load 200 μL of the supernatant into a 96 well clear plate.

8. Read plate at 525 nm (absorbance) in endpoint mode.

9. Calculate specific activity:

     *Adjusted for Enzyme Control
     **Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).

Per Well:

• rhTGM1: 1.6 μg

• Substrate: 10 mM

Background: Transglutaminase 1/TGM1

Transglutaminase 1, also known as TGM1, Transglutaminase K, keratinocyte transglutaminase, or epidermal TGase, is a member of the transglutaminase family. Transglutaminases catalyze the calcium-dependent formation of isopeptide bonds between lysine and glutamine residues (1). TGM1 plays an important role in formation of the cornified envelope of differentiating keratinocytes in the epidermis through its cross-linking of epidermal proteins (2). The majority of cases of lamellar ichthyosis result from mutations present within the TGM1 gene and the subsequent dysfunction of TGM1 (1, 3).

• References:

1. Lorand, L. and R.M. Graham (2003) Nature Rev. Mol. Cell. Biol. 4:140.

2. Eckert, R.L. et al. (2005) J. Invest. Dermatol. 124:481.

3. Farasat, S. et al. (2009) J. Med. Genet. 46:103.

• Entrez Gene IDs:

  7051 (Human); 21816 (Mouse); 60335 (Rat)

• Alternate Names:

  Epidermal TGase; ICR2; KTG; LI; LI1; LI1EC 2.3.2.13; protein-glutamine gamma-glutamyltransferase K; protein-glutamine-gamma-glutamyltransferase); TG(K); TGASE; TGase-1; TGK; TGKTGase K; TGM1; transglutaminase 1 (K polypeptide epidermal type I; Transglutaminase 1; Transglutaminase K; transglutaminase, keratinocyte; transglutaminase-1