A sensitive and rapid fluorescence-based assay for determination of tetanus toxin peptidase activity.
The light chain of tetanus toxin (TeNT-L chain), endowed with a zinc metalloendopeptidase activity, cleaves specifically the vesicle-associated membrane protein (VAMP), also called synaptobrevin, at a single peptide bond (Gln76-Phe77), resulting in the blockade of neuroexocytosis. The 50-mer synaptobrevin peptide S 39-88, synthesized by solid-phase peptide synthesis, was determined to be the minimum substrate of TeNT still notably hydrolyzed by TeNT-L chain. In this peptide, Tyr88 was substituted by the highly fluorescent amino acid (L) pyrenylalanine (Pya) which was synthesized in good yields by an enantioselective method. The fluorescent substrate [Pya88] S 39-88 was cleaved four times more rapidly by TeNT-L chain than S 39-88 (kcat/Km = 9635 and 2455 M-1.min-1, respectively). One of the two metabolites formed by the action of TeNT L chain, [Pya88] S 77-88, was easily separated from the substrate in one step using Sep-Pak Vac C18 cartridges and its concentration quantified by fluorescence. This novel enzymatic assay, which could be easily extended to other clostridial neurotoxins, is a major improvement in term of sensitivity and time saving, compared to currently used methods (SDS-PAGE, HPLC). It lends itself readily to automation for large-scale screening of selective and potent inhibitors of these neurotoxins which remain to be developed.
Soleilhac
J M
JM
Department de Pharmacochimie Moléculaire et Structurale, U266 INSERM-URA D1500 CNRS, UFR des Sciences, Pharmaceutiques et Biologiques, Faculté de Pharmacie, Paris, France.
Cornille
F
F
Martin
L
L
Lenoir
C
C
Fournié-Zaluski
M C
MC
Roques
B P
BP
D000595:Amino Acid Sequence; D001681:Biological Assay; D005453:Fluorescence; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000032:analysis* / Q000378:metabolism; D008969:Molecular Sequence Data; D050683:R-SNARE Proteins; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013744:Tetanus Toxin / Q000032:analysis* / Q000378:metabolism
D008565:Membrane Proteins; D050683:R-SNARE Proteins; D013744:Tetanus Toxin; D008666:Metalloendopeptidases