Active Peptidylglycine Alpha Amidating Monooxygenase (PAM)

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 was using Dns-Tyr-Val-Gly as substrate, thus the activity of recombinant human PAM was measured by its ability to hydrolyze Dns-Tyr-Val-Gly to Dns-Tyr-Val-NH2. The reaction was performed in 100 mM MES/KOH pH 6.0, 30 mM KI, 30 mM KCl, 1 uM cupric sulfate, 100 ug/ml catalase (APC418Hu05), 1% (v/v) ethanol, 0.001% (v/v) Triton X-100 and 10 mM ascorbate. 250 ul 0.7 mM substrate of Dns-Tyr-Val-Gly was added with 250 ul various concentrations of recombinant human PAM (1 ug/ml,5 ug/ml, 10 ug/ml and 20 ug/ml). Incubated at 37℃ for 30min, the reaction was stopped by addition 6% (v/v) TCA. The product and substrate was detected by RP-HPLC with UV-detection at 280 nm, the analyses were performed at 25℃ employing a Agilent ZORBAX Poroshell SB C18 column (9.4×250 mm, 5 μm), the flow rate was 1 ml/min. The mobile phase consisted of 100 mM sodium acetate (pH 6.5) and 30 min linear gradient of 10-90% acetonitrile. At 30-35 min, the mobile phase consisted of 90% acetonitrile and 10% sodium acetate. The result was shown in Figure 1. As the Figure 1 shows, the substrate have been hydrolyzed to Dns-Tyr-Val-NH2 after incubating with recombinant human PAM. The retention time of Dns-Tyr-Val-Gly and Dns-Tyr-Val-NH2 is 24.088 and 30.421 respectively (Figure 2). The specific activity of recombinant human PAM is >6600 pmol/min/µg.

Figure 2. The reaction product compared with standard Dns-Tyr-Val-Gly and Dns-Tyr-Val-NH2.

Figure 3. The sandard curve of Dns-Tyr-Val-NH2