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Rat PAM protein (active)

Rat PAM protein (active). GTX00374-pro
Rat PAM protein (active). GTX00374-pro

Cat No. GTX00374-pro

Application

Functional Assay

Species

Rat
Package
10 μg ($349)

APPLICATION

Application Note

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is an enzyme that is required for the biosynthesis of many signaling peptides. It has two enzymatically active domains with catalytic activities-peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). These catalytic domains work sequentially to catalyze neuroendocrine peptides to active alpha-amidated products. A typical activity assay using Dns-Tyr-Val-Gly as substrate, thus the recombinant rat PAM activity was measured by its ability to hydrolyze Dns-Tyr-Val-Gly to Dns-Tyr-Val-NH2. The reaction was preformed in 1mL containing 100 mM MES/KOH (pH 6.0), 30 mM KI, 30 mM KCl, 1μmol/L cupric sulfate, 100 μg/ml catalase, 1% (v/v) ethanol, 0.001% (v/v) Triton X-100, 10 mM ascorbate, 0.35 mM/L Dns-Tyr-Val-Gly (0.2 mg/ml) and initiated by addition various concentrations of PAM (0.1 μg/ml, 1 μg/ml, 5 μg/ml). Incubated at 37ºC for 30min, the reaction stopped by addition 6% (v/v) TFA. The product and substrate was detected by RP-HPLC with UV-detection at 280nm, the analyses were performed at 25ºC employing a Agilent ZORBAX Poroshell SB C18 column (9.4×250mm, 5μm), the flow rate was 1 ml/min. The mobile phase consisited of 100 mM sodium acetate (pH 6.5) and 35min linear gradient of 10-90% acetonitrile. The reaction product compared with standard Dns-Tyr-Val-Gly and Dns-Tyr-Val-NH2. After 30min later, the substrate have been hydrolyzed when the PAM was 5μg/ml. The retention time of Dns-Tyr-Val-Gly and Dns-Tyr-Val-NH2 is 24.315 and 30.806 respectively.

Observed MW

88 kDa.

PROPERTIES

Form

Lyophilized powder

Buffer

Lyophilized from PBS (pH 7.4) with 0.01% SKL, 1 mM DTT, 5% Trehalose, Proclin300. Reconstitute with 10 mM PBS (pH 7.4) to a concentration of 0.1-1.0 mg/mL. Do not vortex

Storage

For short-term storage (1-2 weeks), store at 4ºC. For long-term storage, store at -20ºC or below. After reconstitution, keep as concentrated solution.Avoid freeze-thaw cycles.

Region/Sequence

N-terminal His-Tag; Phe36~Val820 (NP_037132.2)

Expression System

293F cell

Purity

> 95%

Endotoxin

< 1 EU/μg

Conjugation

Unconjugated

Note

For laboratory use only. Not for any clinical, therapeutic, or diagnostic use in humans or animals. Not for animal or human consumption.

TARGET

Synonyms

peptidylglycine alpha-amidating monooxygenase , PHM

Background

catalyzes conversion of glycine-extended peptides into biologically active alpha-amidated peptides [RGD, Feb 2006]

Database

Research Area

DATA IMAGES

Rat PAM protein (active). GTX00374-pro

GTX00374-pro Functional Assay Image

Hydrolysis assay of Dns-Tyr-Val-Gly by GTX00374-pro Rat PAM protein (active) for 30 min. The substrate (Dns-Tyr-Val-Gly) have been hydrolyzed to product (Dns-Tyr-Val-NH2) when the Rat PAM protein (active) was 5 μg/ml. The retention time of Substrate and Product are 24.315 and 30.806, respectively.
Upper: Activity assay of Rat PAM protein (active) by HPLC.
Lower: The reaction products compared with standard Dns-Tyr-Val-Gly and Dns-Tyr-Val-NH2.

Rat PAM protein (active). GTX00374-pro

GTX00374-pro Image

SDS-PAGE analysis of GTX00374-pro Rat PAM protein (active).

REFERENCE

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REVIEW

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Package List Price ($)
$ 349