L 2 3 4 5 6 3h phenylalanine

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), horse serum, and Penicillin-Streptomycin solution were purchased from Gibco-Thermo-Fisher Scientific (Grand Island, NY, USA). L-[2,3,4,5,6-3H] Phenylalanine and L-[Ring-3, 5-3H]-Tyrosine was purchased from Perkin-Elmer (Waltham, MA, USA), Prune extract-60% enriched polyphenol extract (PE60) was purchased from PL Thomas (Morristown, NJ, USA). All other chemicals were of reagent grade, and were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

For the cell‐free synthesis of radioligands one amino acid was replaced with a tritiated variant depending on its abundance in the respective ligand. Complement 5a was produced in the presence of 1 μm (FM)/2.1 μm (RM) L‐[2,3‐³H]‐proline (specific activity 55 Ci·mmol⁻¹, Perkin Elmer, Boston, USA). For labelling of endothelin‐1 1 μm (FM)/0.9 μm (RM) L‐[2,3,4,5,6‐³H]‐phenylalanine (specific activity 128.1 Ci·mmol⁻¹, Perkin Elmer) was added. The cell‐free reaction was initiated omitting the labelled amino acid and incubated at 30 °C, 100 rpm for 3 h. The reaction mix was removed from the D‐tube^™ dialyzer and incubated with 0.2 mL Ni‐NTA magnetic agarose beads suspension (Qiagen) per mL reaction mix for 10 min to remove undesired side products translated with endogenously present amino acids from the S30 extract. The reaction mix was removed from the magnetic beads, supplemented with 0.25 mm L‐histidine to account for potential losses in the IMAC (immobilized metal ion affinity chromatography) step and the tritium‐labelled amino acid. The feeding mix was discarded and replaced with a fresh mix containing the labelled amino acid. Reactions were incubated at 30 °C in a water bath under constant shaking overnight.