A fluorescent substrate of transglutaminase for detection and characterization of glutamine acceptor compounds.
A fluorescent dipeptide was designed to discover glutamine acceptor proteins of transglutaminase. Starting materials for synthesis were the commercially available compounds carbobenzoxy-L-glutaminylglycine (CBZ-Gln-Gly) and monodansylcadaverine (C-DNS) which were coupled to obtain CBZ-Gln-Gly-C-DNS 1 [1-N-(carbobenzoxy-L-glutaminylglycyl)-5-N- (5'-N', N'-dimethylamino-1'-naphthalenesulfonyl)- diamidopentane]. The glutamine peptide is a substrate of bacterial transglutaminase from Streptoverticillium mobaraense as well as of the guinea pig liver enzyme. This could be shown by incorporating 1 into alpha s1-casein resulting in a significant increase in fluorescence intensity and a concomitant inhibition of cross-linking reaction. Additionally, dipeptide 1 is a useful tool to characterize the specificity of transglutaminase toward small primary amines. We established a sensitive HPLC assay and determined the kinetic parameters of several alkylamines. Hydrolysis of 1 is suppressed in the presence of the nucleophiles as it could be demonstrated with different concentrations of butylamine in semiquantitative studies. Together with labeled primary amines, reagent 1 seems to be a particularly suitable tool for examining acceptor-donor relationships of transglutaminase substrates.
Pasternack
R
R
Fachbereich Chemische Technologie, Fachhochschule Darmstadt, Germany.
Laurent
H P
HP
Rüth
T
T
Kaiser
A
A
Schön
N
N
Fuchsbauer
H L
HL
D004151:Dipeptides / Q000138:chemical synthesis*; D005456:Fluorescent Dyes; D005973:Glutamine / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D015394:Molecular Structure; D013306:Streptomycetaceae / Q000201:enzymology; D013449:Sulfonamides / Q000138:chemical synthesis*; D011503:Transglutaminases / Q000737:chemistry*
C108034:1-N-(carbobenzoxy-glutaminylglycyl)-5-N-(5'-N',N'-dimethylamino-1'-naphthalenesulfonyl)diamidopentane; D004151:Dipeptides; D005456:Fluorescent Dyes; D013449:Sulfonamides; D005973:Glutamine; D011503:Transglutaminases