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 performed in 1ml containing 100mM MES/KOH pH 6.0, 30mM KI, 30mM KCl, 1μmol/L cupric sulfate, 100ug/ml catalase, 1% (v/v) ethanol,0.001% (v/v) Triton X-100, 10mM ascorbate ,0.35mM/L Dns-Tyr-Val-Gly(0.2mg/ml) and initiated by addition various concentrations of PAM(0.1ug/ml,1ug/ml,5ug/ml). Incubated at 37℃ for 30min, the reaction stopped by addition 6% (v/v)TCA . The product and substrate was detected by RP-HPLC with UV-detection at 280nm, the analyses were performed at 25℃ employing a Agilent ZORBAX Poroshell SB C18 column(9.4×250mm,5μm), the flow rate was 1ml/min. The mobile phase consisited of 100 mM sodium acetate (pH 6.5) and 35min linear gradient of 10-90% acetonitrile. The result was shown in Figure 1. As the Figure 1 shows, after 30min later, the substrate have been hydrolyzed when the PAM was 5 ug/ml. The retention time of Dns-Tyr-Val-Gly and Dns-Tyr-Val-NH2 is 24.315 and 30.806 respectively. The specific activity of recombinant rat PAM is > 7600 pmol/min/μg.

Specific Activity (pmol/min/μg) =
?*Adjusted for Substrate Blank
?**Derived using calibration standard Dns-Tyr-Val-NH2