Recombinant Human Methionine Aminopeptidase 1D Protein, CF

• Reactivity: Hu
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant Human Methionine Aminopeptidase 1D Protein, CF Summary

• Details of Functionality: Measure by its ability to remove methionine from a fluorogenic peptide substrate H-Met-Gly-Pro-AMC (Catalog # ES017). The resulting GP-AMC is cleaved by Recombinant Human DPPIV/CD26 (Catalog # 9168-SE). The specific activity is >30 pmol/min/µg, as measured under the described conditions.
• Source: Spodoptera frugiperda, Sf 21 (baculovirus)-derived human Methionine Aminopeptidase 1D/MAP1D protein
  Arg44-Ala335, with an N-terminal Met and a C-terminal 10-His tag
• Accession #: Q6UB28
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Gene: METAP1D
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 34 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 38 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by silver stain.
• Assay Procedure:
  • Activation Buffer: 50 mM HEPES, 0.1 mM CoCl₂, 100 mM NaCl, pH 7.5
  • Assay Buffer: 25 mM Tris, pH 8.0
  • Recombinant Human Methionine Aminopeptidase 1D/MAP1D (rhMAP1D) (Catalog # 4714-ZN)
  • Recombinant Human DPPIV/CD26 (rhDPPIV/CD26) (Catalog # 9168-SE)
  • Substrate: H-Met-Gly-Pro-AMC (Catalog # ES017)
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rhMAP1D to 40 µg/mL in Activation Buffer.
  2. Dilute Substrate to 500 µM with Activation Buffer.
  3. Combine equal volumes of 40 µg/mL rhMAP1D with 500 µM Substrate. Include a Substrate Blank containing Activation Buffer and 500 µM Substrate.
  4. Incubate at room temperature for 1 hour in the dark.
  5. Stop the reaction by heating for 5 minutes at 95 °C. Put on ice for 3 minutes. Centrifuge briefly in a microcentrifuge.
  6. Dilute rhDPPIV/CD26 to 2 ng/µL in Assay Buffer.
  7. Load 50 µL of 2 ng/µL rhDPPIV/CD26 into a plate, and add 50 µL of the rhMAP1D/Substrate mixture (include Substrate Blank).
  8. Incubate plate at room temperature for 15 minutes in the dark.
  9. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in endpoint mode.
  10. Calculate specific activity:

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

  *Adjusted for Substrate Blank

  **Derived using calibration standard 7-amino, 4-Methyl Coumarin (AMC) (Sigma, Catalog # A-9891).

  Per Well:
  • rhMAP1D: 1 µg
  • rhDPPIV/CD26: 0.1 µg
  • Substrate: 125 µM

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Methionine Aminopeptidase 1D Protein, CF

• EC 3.4.11.18
• MAP1D
• MAP1DCDS of metAP-3 within PCR fragment
• MetAP1D
• Metap1l
• Methionine Aminopeptidase 1D
• methionine aminopeptidase 1D, mitochondrial
• methionyl aminopeptidase type 1D (mitochondrial)
• Methionyl aminopeptidase type 1D, mitochondrial

Background

Methionine Aminopeptidase 1D (MAP1D) is a member of the Methionine Aminopeptidase family (MetAPs) (1). MetAPs catalyze the removal of the initiator methionine residue from nascent peptides (2) and are essential for cell growth (3). MAP1D is localized in mitochondria (4) and is over-expressed in colon cancer cell lines and colon tumors (1). The N-terminal leader peptide (residue 1-43) of MAP1D has been shown to be responsible for targeting to mitochondria (4).

1. Leszczyniecka, M. et al. (2006) Oncogene 25:3471.
2. Lowther, W.T. and B.W. Matthews (2000) Biochim. Biophys. Acta. 1477:157.
3. Li, X. and Y.H. Chang (1995) Proc. Natl. Acad. Sci. USA 92:12357.
4. Serero, A. et al. (2003) J. Biol. Chem. 278:52953.

Bioinformatics

• Gene Symbol: METAP1D
• Uniprot:
  • Q6UB28()