Recombinant Mouse BLMH/Bleomycin Hydrolase Protein, CF

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

Recombinant Mouse BLMH/Bleomycin Hydrolase Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze Met-AMC. The specific activity is >1,500 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived mouse BLMH/Bleomycin Hydrolase protein
  Asn2-Glu455 with an N-terminal Met and 6-His tag
• Accession #: Q8R016
• N-terminal Sequence: Met
• Protein/Peptide Type: Recombinant Enzymes
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 53 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: 45-55 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -70 °C as supplied.
  • 3 months, -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris, NaCl, Glycerol and DTT.
• Purity: >95%, by SDS-PAGE visualized with Silver Staining and quantitative densitometry by Coomassie® Blue Staining.
• Assay Procedure:
  • Assay Buffer: 50 mM HEPES, 5 mM EDTA, 10 mM DTT, pH 7.0
  • Recombinant Mouse BLMH/Bleomycin Hydrolase (rmBLMH) (Catalog # 6180-CY)
  • Substrate: Met-AMC (Bachem, Catalog # I-1265), 100 mM stock in DMSO
  • F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
  • Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
  1. Dilute rmBLMH to 10 µg/mL in Assay Buffer.
  2. Incubate at 37 °C for 30 minutes.
  3. Dilute Activated rmBLMH to 0.4 µg/mL.
  4. Dilute Substrate to 2 mM in Assay Buffer.
  5. Load 50 µL of the 0.4 µg/mL rmBLMH into a plate, and start the reaction by adding 50 µL of 2 mM Substrate. Include a Substrate Blank containing 50 µL Assay Buffer and 50 µL of 2 mM Substrate without any rmBLMH.
  6. Read at excitation and emission wavelengths of 380 nm and 460 nm (top read), respectively, in kinetic mode for 5 minutes.
  7. 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 # A-9891).

  Per Well:
  • rmBLMH: 0.02 µg
  • Substrate: 1 mM

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.


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

Alternate Names for Recombinant Mouse BLMH/Bleomycin Hydrolase Protein, CF

• BHBMHBLM hydrolase
• bleomycin hydrolase
• BLMH
• BMH
• EC 3.4.22.40

Background

Bleomycin hydrolase (BLMH) is a cysteine peptidase of the papain superfamily. It is named for its ability to hydrolyze the anti‑tumor agent bleomycin and inactivate it (1). It has a papain-like catalytic triad (Cys-His-Asp) with optimum activity at neutral pH. In mammals it is expressed ubiquitously in all types of tissues and its expression is up‑regulated in many tumors. It is present in the cytoplasm as homohexameric protein of approximately 300 kDa. In addition to its aminopeptidase activity, it has homocysteine-thiolactonase activity. The normal physiological function of BLMH is not clear. BLMH inactivates bleomycin, a glycopeptide anti‑cancer agent, by deaminating it (2). BLMH has been suggested to play a role in the generation of MHC class I-presented peptides (3, 4). Diminished BLMH activity may contribute to the pathology of Alzheimer’s disease (AD) (5, 6). It is inhibited by cysteine protease inhibitors such as N‑ethylmaleimide, iodoacetamide, para‑hydroxymercuribenzoate, and E-64.

1. Joshua-Tor, L. and S. A. Johnson (2004) in Handbook of Proteolytic Enzymes, Barrett, A. J. et al. eds. pp. 1197.
2. Schwartz, D. R. et al. (1999) Proc. Natl. Acad. Sci. USA, 96:4680.
3. Kim, E. et al. (2009) J. Immunol. 183:7379.
4. Towne, C. F. et al. (2007) J. Immunol. 178:6923.
5. Suszynska, J. et al. (2010) J. Alzheimers Dis. 19:1177.
6. Lefterov, I. M. et al. (2000) FASEB J. 14:1837.

Bioinformatics

• Uniprot:
  • Q8R016()