• Purity

  >85%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie? Blue Staining.

• Endotoxin Level

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

• Activity

  Measure by its ability to remove methionine from a fluorogenic peptide substrate H-Met-Gly-Pro-AMC (Catalog # ). The resulting GP-AMC is cleaved by Recombinant Human DPPIV/CD26 (Catalog # ). The specific activity is >0.5 pmol/min/μg, as measured under the described conditions. See Activity Assay Protocol on .

• Source

  E. coli-derived Ala2-Glu264, with a C-terminal 10-His tag

• Accession #

• N-terminal Sequence    
  Analysis

  Ala2

• Predicted Molecular Mass

  31 kDa

• SDS-PAGE

  36 kDa, reducing conditions

Assay Procedure

Materials

• Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂, 0.1 M NaCl, pH 7.5

• Assay Buffer: 25 mM Tris, pH 8.0

• Recombinant E. coli Methionine Aminopeptidase (reMAP) (Catalog # 1628-ZN)

• Recombinant Human DPPIV/CD26 (rhDPPIV) (Catalog # )

• Substrate: H-Met-Gly-Pro-AMC (Catalog # )

• F16 Black Maxisorp Plate (Nunc Catalog # 475515)

• Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent

1. Dilute reMAP to 40 μg/mL in Activation Buffer.

2. Dilute Substrate to 200 μM in Activation Buffer.

3. Mix 100 μL of 40 μg/mL reMAP with 100 μL 200 μM Substrate. Include a Substrate Blank containing 100 μL of Activation Buffer and 100 μL 200 μM Substrate.

4. Incubate at 37 °C for 15 minutes.

5. Stop the reaction by heating for 5 minutes at 95 to 100 °C.

6. Place reactions on ice for 3 minutes, then spin briefly.

7. Dilute rhDPPIV/CD26 to 2 μg/mL in Assay Buffer.

8. Load 50 μL of reaction mixture into a plate and add 50 μL of 2 μg/mL rhDPPIV/CD26.

9. Incubate plate with shaking for 10 minutes at room temperature.

10. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively in endpoint mode.

11. Calculate specific activity:

    Specific Activity (pmol/min/μg) =	Adjusted Vmax* (RFU/min) x Conversion Factor** (pmol/RFU)
    	amount of enzyme (μg)

         *Adjusted for Substrate Blank

         **Derived using calibration standard 7-Amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A9891).

Per Well:

• reMAP: 1 μg

• rhDPPIV/CD26: 0.100 μg

• Substrate: 50 μM

Background: Methionine Aminopeptidase

Methionine Aminopeptidase (METAP) carries out the important reaction of removing the initiator Met residue from nascent proteins. There are two types of METAP (1, 2). Type Ia and type IIa are found in eubacteria and archaea, respectively. Type Ib and type IIb are present in eukaryotes and contain additional N-terminal domains. As a metalloenzyme, METAP activity can be inhibited by EDTA. However, the exact identity of the metal ion remains to be uncovered since several metal ions including cobalt, zinc and iron have been suggested (3). 

• References:

1. Bradshaw, R.A. et al. (1998) Trends Biochem. Sci. 23:263.

2. Lowether, W.T. and B.W. Matthews (2000) Biochim. Biophys. Acta 1477:157.

3. Li, J-Y. et al. (2003) Biochem. Biophys. Res. Comm. 307:172.

• Entrez Gene IDs:

  947882 (E. coli)

• Alternate Names:

  Methionine Aminopeptidase