Carboxypeptidase M/CPM Protein, Human (HEK293, His)

Carboxypeptidase M Protein, Human (HEK293, His) is an approximately 55.0 kDa Carboxypeptidase M protein with a His-flag. Carboxypeptidase M is a membrane-bound arginine/lysine carboxypeptidase enzyme belongs to the carboxypeptidases family^[1].

Carboxypeptidase M is a membrane-bound arginine/lysine carboxypeptidase enzyme, cleaves C-terminal basic amino acids with a neutral pH optimum^[1].
  Carboxypeptidase M belongs to the carboxypeptidases family. Carboxypeptidase M is widely distributed in a variety of tissues and cells, and it constitutively expresses in an active form at the surface of specialized cells and tissues in the human body^[2].
Carboxypeptidase M plays an important role in the physiological processes of blood coagulation/fibrinolysis, inflammation, food digestion and pro-hormone and neuropeptide processing^[3].

Measured by its ability to release L-arginine from Benzoyl-Ala-Arg, with detection of the arginine amino group by o-phthaldialdehyde. The specific activity is 187829.19 pmol/min/μg that incubate for 10 minutes at room temperature.

Materials
Assay buffer: 50 mM MES, 5 mM CaCl₂, 0.2% Triton X-100, pH 6.0
Carboxypeptidase M/CPM Protein, Human (HEK293, His) (HY-P7737)
Substrate: Bz-Ala-Arg-OH (HY-P4522), 50 mM in DMSO
Standard: L-Arginine
NaOH, dissolved in distilled water to 2 M
o-Phthaldialdehyde (OPA), dissolved in DMSO to 0.373 M
2-Mercaptoethanol (HY-Y0326)

Procedure
1. Dissolve standards in assay buffer to concentrations of 31250, 15625, 7812, 3906, 1953, 976, 488, 244, 122, 61, 30.5, 15.25, and 0 pmoL. Add an equal volume of 0.2 M NaOH solution containing 15 mM OPA (with 0.1% (v/v) 2-mercaptoethanol) to each concentration gradient. Incubate at room temperature for 10 minutes, then read at excitation wavelength 330 nm and emission wavelength 450 nm.
2. Dilute the Human CPM to 0.04 μg/mL using assay buffer.
3. Dilute the substrate to 1 mM using assay buffer.
4. Mix 150 μL of 0.04 μg/mL test protein with 150 μL of 1 mM substrate to achieve final concentrations of 0.02 μg/mL and 500 μM, respectively. For the control group, add only 150 μL of 1 mM substrate.
5. Incubate at room temperature for 10 minutes.
6. Stop the reaction by adding 300 μL of 0.2 M NaOH solution containing 15 mM OPA (with 0.1% (v/v) 2-mercaptoethanol).
7. After stopping the reaction, add 150 μL of 0.04 μg/mL Human CPM to the control group.
8. Incubate at room temperature for 10 minutes.
9. Add 200 μL of the incubated sample to the plate.
10. Read in endpoint mode at excitation and emission wavelengths of 330 nm and 450 nm.
11. Calculate specific activity:

Human

HEK293

C-6*His

P14384 (L18-D421)

1368  [NCBI]

Partial

LDFNYHRQEGMEAFLKTVAQNYSSVTHLHSIGKSVKGRNLWVLVVGRFPKEHRIGIPEFKYVANMHGDETVGRELLLHLIDYLVTSDGKDPEITNLINSTRIHIMPSMNPDGFEAVKKPDCYYSIGRENYNQYDLNRNFPDAFEYNNVSRQPETVAVMKWLKTETFVLSANLHGGALVASYPFDNGVQATGALYSRSLTPDDDVFQYLAHTYASRNPNMKKGDECKNKMNFPNGVTNGYSWYPLQGGMQDYNYIWAQCFEITLELSCCKYPREEKLPSFWNNNKASLIEYIKQVHLGVKGQVFDQNGNPLPNVIVEVQDRKHICPYRTNKYGEYYLLLLPGSYIINVTVPGHDPHITKVIIPEKSQNFSALKKDILLPFQGQLDSIPVSNPSCPMIPLYRNLPD

Approximately 55.0 kDa

• 
  ≥ 95%, as determined by reducing SDS-PAGE.

Solution.

Supplied as a 0.22 μm filtered solution of 20 mM Tris-HCl, 150 mM NaCl, 1 mM ZnCl₂, pH7.5.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.

• [1]. A Nagae, et al. Carboxypeptidase M in brain and peripheral nerves. J Neurochem  [Content Brief]

  [2]. Kathleen Deiteren, et al. Carboxypeptidase M: Multiple alliances and unknown partners. Clin Chim Acta. 2009 Jan;399(1-2):24-39.  [Content Brief]

  [3]. David Reverter, Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity. J Mol Biol  [Content Brief]