C-terminal α-amidation is the final and essential step in the biosynthesis of several peptide hormones. Peptidylglycine α-amidating monooxygenase (PAM) is the only known enzyme to catalyse this reaction. PAM amidating activity (AMA) is known to be present in human circulation, but its physiological role and significance as a clinical biomarker remains unclear. We developed a PAM-specific amidation assay that utilizes the naturally occurring substrate Adrenomedullin-Gly (ADM-Gly, 1–53). Using our amidation assay we quantified serum amidating activities in a large population-based cohort of more than 4900 individuals. A correlation of serum amidating activity with several clinical parameters including high blood pressure was observed.
Fig: Schematic representation of the AMA Assay. Step 1—amidation: calibrators, controls and samples are dispensed in double determination as indicated (yellow) into a non-binding 96-well plate. The reaction is started by addition of reaction buffer to calibrators, controls and samples (green) and a t = 0 min time-point is generated immediately by transferring equal volumes of each double determination into EDTA prefilled wells (red). The generated t = 0 min time-points are single determinations. After incubation at 37 °C for 40 min an inactivation of remaining samples with EDTA is performed. Step 2—product quantification: following 37 °C incubation and inactivation, all samples are transferred into the sphingotest bio-ADM Assay 96-well immunoluminometric assay (ILMA) plate. The bio-ADM assay is processed according to manufacturer’s specifications. Both, ADM-Gly and bio-ADM are bound by the solid-phase antibody (a). The MACN labelled tracer-antibody (b) specifically recognizes the c-terminally amidated bio-ADM and does not react with ADM-Gly. Following incubation, unbound tracer-antibody is washed away and bio-ADM specific luminescence signals are detected using a standard plate luminometer. The figure was created with Microsoft PowerPoint 2016, version 2105.
Increasing PAM-AMA was an independent predictor of hard outcomes related to hemodynamic stress such as cardiovascular mortality, atrial fibrillation and heart failure during long-term follow-up (8.8 ± 2.5 years). Moreover, results from an animal study in rats utilizing recombinant human PAM provide novel insights into the physiological role of circulating PAM and show its potential significance in circulating peptide amidation.
Kaufmann, P., Bergmann, A. & Melander, O. Novel insights into peptide amidation and amidating activity in the human circulation. Sci Rep 11, 15791 (2021). https://doi.org/10.1038/s41598-021-95305-y