Recombinant Human Histamine N-Methyltransferase/HNMT, CF

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

Recombinant Human Histamine N-Methyltransferase/HNMT, CF Summary

• Details of Functionality: Measured by its ability to methylate histamine. The specific activity is >425 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived human Histamine N-Methyltransferase/HNMT protein
  Ala2-Ala292, with an N-terminal HM and 6-His tag.
• Accession #: P50135
• N-terminal Sequence: His (of HM) and Ser7
• Protein/Peptide Type: Recombinant Enzymes
• Gene: HNMT
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <0.10 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: 32-36 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 and Glycerol.
• Purity: >90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 20 mM Tris, 0.02% Brij-35, pH 8.0
  • Recombinant Human Histamine N-Methyltransferase/HNMT (rhHNMT) (Catalog # 7637-MT)
  • Glutathione, reduced (Amresco, Catalog # 399), 250 mM stock in deionized water
  • Recombinant Human Adenosylhomocysteinase/AHCY (rhAHCY) (Catalog # 6466-AH)
  • Recombinant Human Adenosine Deaminase/ADA (rhADA) (Catalog # 7048-AD)
  • S-adenosylmethionine (Sigma, Catalog # A7007), 10 mM stock in 50% DMSO in deionized water
  • Histamine dichloride (Sigma, Catalog # H7250), 20 mM stock in deionized water
  • ThioGlo® 3 Fluorescent Thiol Reagent (Covalent Associates, Inc., Catalog # T-003), 10 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 the stock of reduced glutathione to 40 µM (40 pmol/µL) in Assay Buffer. This is the first point of the standard curve.
  2. Continue standard curve by performing six ½ serial dilutions of the 40 µM glutathione in Assay Buffer. The standard curve has a range of 31.25 to 2000 pmol per well.
  3. Dilute rhHNMT to 2 µg/mL in Assay Buffer containing 800 µM S-adenosylmethionine.
  4. Dilute rhAHCY to 80 µg/mL in Assay Buffer containing 4 µg/mL rhADA.
  5. To form reaction mixture, combine equal volumes of rhHNMT mixture and rhAHCY mixture. As a control, combine equal volumes of rhAHCY mixture with 800 µM S-adenosylmethionine.
  6. Dilute Histamine to 100 µM.
  7. Load 50 µL of each point of the standard curve into a plate. Include a curve blank containing 50 µL of Assay Buffer.
  8. Load 25 µL of reaction mixture to wells. Start reaction by adding 25 µL of 100 µM Histamine.
  9. Cover plate and incubate at 37°C for 30 minutes.
  10. Dilute ThioGlo® to 100 µM in DMSO.
  11. After incubation, add 50 µL of 100 µM ThioGlo® to each well.
  12. Incubate at room temperature for 5 minutes in the dark.
  13. Read the plate in endpoint mode at excitation and emission wavelengths of 380 nm and 445 nm, respectively.
  14. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Thiol Produced* (pmol)) / (Incubation time (min) x amount of enzyme (µg))

  *Derived from the reduced glutathione standard curve using linear fitting and adjusted for Control.

  Per Well:
  • rhHNMT: 0.025 µg
  • rhAHCY: 1 µg
  • rhADA: 0.050 µg
  • S-adenosylmethionine: 100 µM
  • Histamine: 25 µM
  • ThioGlo®: 50 µM

Notes

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

Alternate Names for Recombinant Human Histamine N-Methyltransferase/HNMT, CF

• EC 2.1.1.8
• Histamine NMethyltransferase
• Histamine N-Methyltransferase
• HMT
• HNMT
• HNMT-S1
• HNMT-S2

Background

Histamine N-Methyltransferase (HNMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine to the N_e2 atom of the imidazole ring in histamine producing methylhistamine (1). Because histamine is a potent mediator of numerous biological events, including the development of allergic inflammation, termination of its action by N-methylation is important event. Two major pathways exist for the inactivation of histamine in mammals (2). The pathway of histamine inactivation through HNMT plays a major role in bronchial epithelial and endothelial cells of the human airways and gut and HNMT is the only enzyme responsible for termination of the neurotransmitter action of histamine in the brain. The human HNMT gene contains a common single nucleotide polymorphism (SNP) that codes for either threonine or isoleucine at residue 105 (3). The 105I variant has decreased activity and lower protein levels relative to 105T. This SNP does not pose a serious risk for histamine accumulation (4).

1. Francis, D. M. (1980) Biochem. J. 187:819.
2. Maints, L. and Novak, N. (2007) Am. J. Clin. Nutr. 85:1185.
3. Rutherford. K. et al. (2008) Biochemistry. 47:893.
4. Garcia-Martin, E. et al. (2009) Pharmacogenomics 10:867.

Publications for Histamine N-Methyltransferase/HNMT (7637-MT)(1)

Publication using 7637-MT

• HH Maw, X Zeng, S Campbell, ME Taub, AM Teitelbaum N-methylation of BI 187004 by thiol S-methyltransferase Drug Metab. Dispos., 2018-03-07;0(0):. 2018-03-07 [PMID: 29514826] (Bioassay, Human)

Bioinformatics

• Gene Symbol: HNMT
• Uniprot:
  • P50135(Human)