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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Synthesis and Bioactivity Studies on the C-TerminallyExpressed Heptapeptide Orthologues of VariousProenkephalin A SequencesFruzsina Babos 1,3 , Engin Bojnik 2 , Sándor Benyhe 2 , and Anna Magyar 31 Department of Organic Chemistry, Eötvös Lorand University, Budapest, Hungary;2 Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences,Szeged, Hungary; 3 Research Group of Peptide Chemistry, Hungarian Academy of Sciences,Eötvös Lorand University, Budapest, HungaryIntroductionSince the isolation of enkephalins a number of other opioid peptides have been discovered,including heptapeptide with the sequence of Tyr-Gly-Gly-Phe-Met-Arg-Phe (Metenkephalin-Arg6-Phe7,YGGFMRF or MERF). The heptapeptide MERF is a potent opioidcleft from the sequence of proenkephalin A (PENK), the common precursor of Met- (ME)and Leu-enkephalin (LE).Our bioinformatic analysis exposed chemical biodiversity at the heptapeptide region ofPENK among 56 animals [1]. Four novel orthologoues sequences were found, such asYGGFMGY (Zebrafish), YGGFMRY (Newt), YGGFMKF (Hedgehog tenrek) andYGGFMRI (Mudpuppy).Each novel heptapeptides were subjected to functionality studies, using receptorbinding and G-protein activation assays. The relative affinities of the heptapeptides revealrather mu-receptor preference over the delta-receptors. [ 35 S]GTPgS assay has demonstratedthat these novel heptapeptides are also potent in stimulating the regulatory G-proteins.Results and DiscussionThe peptides were synthesized by automated SPPS (SYRO, Multisyntech), using Fmoc/tBustrategy on 2-chloro-trityl resin with double-coupling protocol. The crude products werepurified by HPLC. The structure of the peptides was proved by electron spray ionization(ESI) mass spectrometry.All binding assays wereperformed at 25°C for 30minutes in 50 mM Tris–HClbuffer (pH 7.4) in a finalvolume of 1 ml, containing 1mg BSA and 0.2–0.4 mg/mlmembrane protein.Rat brain membranefractions (~10 µg of protein/sample) were incubated at30°C for 60 min in Tris–EGTA buffer (50 mM Tris–HCl, 1 mM EGTA, 3 mMMgCl 2 , 100 mM NaCl, pH7.4) containing [ 35 S]GTPγS(0.05 nM) and increasing (10 -9 to 10 -5 M) concentrations ofthe compounds tested in thepresence of 30 µM GDP in afinal volume of 1 ml. TotalFig. 1. Receptor binding and G-Protein activation assays.binding was measured in thein the presence of 10 µMunlabeled GTPγS and subtracted from total binding to calculate the specific binding. Alldata are expressed as means ± standard error of the mean of n experiments. Curve fittingwas performed using PRISM 4.0 (GraphPad Software Inc., San Diego, U.S.A.).424