Recombinant Human PAM Protein, CF
Recombinant Human PAM Protein, CF Summary
Met1-Val817, with a C-terminal 10-His tag
CF stands for Carrier Free (CF). We typically add Bovine Serum Albumin (BSA) as a carrier protein to our recombinant proteins. Adding a carrier protein enhances protein stability, increases shelf-life, and allows the recombinant protein to be stored at a more dilute concentration. The carrier free version does not contain BSA.
In general, we advise purchasing the recombinant protein with BSA for use in cell or tissue culture, or as an ELISA standard. In contrast, the carrier free protein is recommended for applications, in which the presence of BSA could interfere.
|Formulation||Supplied as a 0.2 μm filtered solution in Tris and NaCl.|
|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.
- Assay Buffer: 50 mM MES, pH 6.0
- Recombinant Human Peptidylglycine alpha ‑Amidating Monooxygenase/PAM (rhPAM) (Catalog # 4837-AM)
- Substrate: Sodium Hippurate [0.5 M Hippuric Acid (Sigma, Catalog # 112003) in 0.5 M NaOH]
- Standard: Sodium Glyoxylate
- Glyoxylate Development Solution
- Catalase (Sigma, Catalog # C30), 100,000 units/mL stock in 50 mM MES, pH 6.5
- L-Ascorbic Acid (Sigma, Catalog # 255564), 0.5 M stock in deionized water
- Copper (II) Chloride (Sigma, Catalog # 222011), 1 M stock in deionized water
- F16 Black Maxisorp Plate (Nunc, Catalog # 475515)
- Fluorescent Plate Reader (Model: SpectraMax Gemini EM by Molecular Devices) or equivalent
- Dilute the standard to 2000 μM in Assay Buffer. Prepare the standard curve by performing six ½ serial dilutions of the 2000 μM standard. The standard curve has a range of 0.625 to 40 nmol per well.
- Dilute rhPAM to 5 ug/mL in cold Assay Buffer. Keep on ice.
- Dilute Substrate to 20 mM in Assay Buffer.
- Prepare a Reaction Mixture containing 4000 Units/mL of Catalase, 0.2 μM of Copper Chloride and 10 mM of Ascorbic Acid in cold Assay Buffer. Keep on ice. (Note: Dilute Copper Chloride to 10 μM in deionized water before adding to the mixture to prevent precipitation.)
- In microtubes, combine 75 μL of rhPAM with 150 μL of Reaction Mixture. As a Substrate Blank, combine 75 μL of Assay Buffer with 150 μL of Reaction Mixture. For the standard curve, combine 75 μL of each dilution of the standard curve with 150 μL of Reaction Mixture.
- Start the reaction by adding 75 μL of 20 mM Substrate to all vials.
- Incubate at 37 °C for 1 hour.
- Add 300 μL of Glyoxylate Development Solution to each vial, mix briefly and incubate at 95‑100 °C for exactly 2 minutes.
- Cool the vials on ice for 3 minutes.
- Into the plate, pipette 160 μL of Substrate Blank, each dilution of the standard curve, and reactions to empty wells in triplicate.
- Read plate in endpoint mode at excitation and emission wavelengths of 330 nm and 415 nm (top read), respectively.
- Calculate specific activity:
Specific Activity (pmol/min/µg) =
|Glyoxylate produced* (nmol) x (1000 pmol/nmol)|
|Incubation time (min) x amount of enzyme (µg)|
*Derived from a sodium glyoxylate standard curve using linear fitting and adjusted for Substrate Blank.Per Reaction:
- rhPAM: 0.1 μg
- Standard Curve: 0.625, 1.25, 2.5, 5, 10, 20, 40 nmol
Pedptidylglycine alpha -Amidating Monooxygenase (PAM) catalyzes the C-terminal amidation that is required for the function of a number of peptide hormones (1). PAM possesses two enzymatic activities on a single polypeptide chain (2), due to the presence of a peptidylglycine alpha ‑hydroxylating monooxygenase (PHM) domain and a peptidyl‑ alpha ‑hydroxyglycine alpha ‑amidating lyase (PAL) domain. The C-terminal glycines of precursor peptides are hydroxylated at the glycine alpha carbon by the PHM activity in a reaction that requires ascorbate, then the PAL activity completes the amidation, releasing glyoxylate in the process. PAM is required for the biosynthesis of peptides such as Substance P, neuropeptide Y, oxytocin, vasopressin, and calcitonin (3). PAM is highly expressed in tissues that synthesize bioactive peptides, such as the thyroid and pituitary glands. The enzyme is generally stored in secretory granules, but soluble secreted forms have been observed (3). Recombinant human PAM was expressed as a C-terminally truncated protein lacking its transmembrane and cytosolic domains to facilitate its secretion.
- Kizer J.S. et al. (1986) Endocrinol. 118:2262.
- Perkins S.N. et al. (1990) Biochem. Biophys. Res. Commun. 171:926.
- Eipper B.A. et al. (1993) Protein Sci. 2:489.
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