Selective labelling of melittin with a fluorescent dansylcadaverine probe using guinea-pig liver transglutaminase.
Melittin, a C-terminal glutamine peptide, incorporated the fluorescent probe monodansylcadaverine (DNC) when catalysed by guinea-pig liver transglutaminase and Ca2+, as determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A 1:1 adduct DNC-melittin was identified in which a single glutamine residue out of two, i.e. Gln25, acts as acyl donor. Incubation of melittin with transglutaminase in the absence of DNC originated high molecular mass complexes indicative that the peptide lysine residue can act as an acyl acceptor. The DNC-melittin was about 3 times more active in the lysis of red cell membranes than native melittin. Fluorescence study of the labelled melittin in the submicromolar range where it is active on cells showed that while totally exposed to solvent in methanol solution, both Trp and dansyl groups are buried in buffer solution. This strongly suggests that DNC-melittin is self-associated and indeed more active than the native melittin in the same conditions.
Pérez-Payá
E
E
Departamento de Bioquímica y Biología Molecular, Universitat de Valencia, Spain.
Thiaudière
E
E
Abad
C
C
Dufourcq
J
J
D000595:Amino Acid Sequence; D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives*; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D006461:Hemolysis; D006801:Humans; D008099:Liver / Q000201:enzymology; D008555:Melitten / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011503:Transglutaminases / Q000378:metabolism*
D005456:Fluorescent Dyes; D008555:Melitten; D011503:Transglutaminases; C008542:monodansylcadaverine; D002103:Cadaverine; D002118:Calcium