Recombinant Human Histamine N-Methyltransferase/HNMT, CF

R&D Systems | Catalog # 7637-MT

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Key Product Details

  • R&D Systems E. coli-derived Recombinant Human Histamine N-Methyltransferase/HNMT (7637-MT)
  • Quality control testing to verify active proteins with lot specific assays by in-house scientists
  • All R&D Systems proteins are covered with a 100% guarantee

Source

E. coli

Accession Number

P50135

Applications

Enzyme Activity
View all Histamine N-Methyltransferase/HNMT Proteins and Enzymes »

Product Specifications

Source

E. coli-derived human Histamine N-Methyltransferase/HNMT protein
Ala2-Ala292, with an N-terminal HM and 6-His tag.

Purity

>90%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane.

Endotoxin Level

<0.10 EU per 1 μg of the protein by the LAL method.

N-terminal Sequence Analysis

His (of HM) and Ser7

Predicted Molecular Mass

34 kDa

SDS-PAGE

32-36 kDa, reducing conditions

Activity

Measured by its ability to methylate histamine.
The specific activity is >425 pmol/min/μg, as measured under the described conditions.

Formulation, Preparation, and Storage

7637-MT
Formulation Supplied as a 0.2 μm filtered solution in Tris, NaCl and Glycerol.
Shipping The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.
Stability & 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.

Background: Histamine N-Methyltransferase/HNMT

Histamine N-Methyltransferase (HNMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine to the Ne2 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).

References

  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.

Long Name

Histamine N-Methyltransferase

Alternate Names

Histamine NMethyltransferase, HMT, HNMT, HNMT-S1, HNMT-S2

Entrez Gene IDs

3176 (Human); 140483 (Mouse); 81676 (Rat)

Gene Symbol

HNMT

UniProt

P50135 (Human)

Additional Histamine N-Methyltransferase/HNMT Products

Product Documents for Recombinant Human Histamine N-Methyltransferase/HNMT, CF

Certificate of Analysis

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Note: Certificate of Analysis not available for kit components.

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Product Specific Notices for Recombinant Human Histamine N-Methyltransferase/HNMT, CF

For research use only

Citations for Recombinant Human Histamine N-Methyltransferase/HNMT, CF

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Protocols

View specific protocols for Recombinant Human Histamine N-Methyltransferase/HNMT, CF (7637-MT):

Materials
  • 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

FAQs

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