• Purity

  >95%, 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 >50 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on www.RnDSystems.com

• Source

  Spodoptera frugiperda,     Sf 21 (baculovirus)-derived Met2-Lys684, with an N-terminal Met and 6-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  No results obtained, Met predicted. Protein identity confirmed by MS analysis of tryptic fragments.

• Predicted Molecular Mass

  78 kDa

• SDS-PAGE

  65-70 kDa, reducing conditions

Assay Procedure

Materials

• Assay Buffer: deionized water

• Recombinant Human Transglutaminase 4/TGM4 (rhTGM4) (Catalog # 5760-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 sodium acetate pH 5.0, 12.5 mM DTT, 200 mM CaCl₂, 125 mM hydroxylamine hydrochloride, and 627 mM NaCl in deionized water.

2. Dilute rhTGM4 to 0.2 mg/mL in Assay Buffer.

3. Mix 30 μL dilute rhTGM4 with 120 μL Substrate Mixture. Include an enzyme blank 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 Substrate Blank
     **Derived using calibration standard L-glutamic acid gamma -monohydroxamate (Sigma, Catalog # G2253).

Per Well:

• rhTGM4: 1.6 μg

• Substrate: 10 mM

• Hydroxylamine: 20 mM

Background: Transglutaminase 4/TGM4

Transglutaminase 4, also known as TGM4, TG4, hTGP, or the prostate-specific transglutaminase, is a member of the transglutaminase family that is exclusively expressed and secreted from the prostate (1, 2). Transglutaminases catalyze the calcium-dependent formation of isopeptide bonds between lysine and glutamine residues (3). In rodents, TGM4 is responsible for the formation of the copulatory plug and sperm antigenicity, but the function in humans is largely unknown (4). The expression of TGM4 is primarily regulated by retinoic acid and secondarily modulated by androgens (5).

• References:

1. Dubbink, H.J. et al. (1996) Biochem. J. 315:901.

2. Dubbink, H.J. et al. (1999) Lab. Invest. 79:141.

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

4. Williams-Ashman, H.G. et al. (1972) Proc. Natl. Acad. Sci. USA 69:2322.

5. Rivera-Gonzalez, G.C. et al. (2012) Nucleic Acids Res. 40:4825.
   
   

• Entrez Gene IDs:

  7047 (Human); 331046 (Mouse); 64679 (Rat)

• Alternate Names:

  EC 2.3.2.13; Fibrinoligase; hTGP; Prostate transglutaminase; Prostate-specific transglutaminase; protein-glutamine gamma-glutamyltransferase 4; TG(P); TGase P; TGase-4; TGM4; TGP; TGPFLJ26776; transglutaminase 4 (prostate); Transglutaminase 4; Transglutaminase P; transglutaminase-4