A sensitive fluorometric assay for tissue transglutaminase.
We have devised a highly sensitive fluorometric well plate assay for tissue transglutaminase that is suitable for multiple kinetic analyses/high-throughput screening of chemical inventories for inhibitors of this enzyme. The procedure measures the rate of fluorescence enhancement (lambda(exc) 260 nm, lambda(em) 538 nm) when 1-N-(carbobenzoxy-l-glutaminylglycyl)-5-N-(5'N'N'-dimethylaminonaphthalenesulfonyl)diamidopentane (glutaminyl substrate) is cross-linked to dansyl cadaverine (amine substrate). The assay procedure can be used to measure the activity of as little as 60 microU of purified guinea pig liver tissue transglutaminase (4.2 ng or 54 fmol of enzyme).
Jeitner
T M
TM
Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York, 10021, USA. tjeitner@burke.org
Fuchsbauer
H L
HL
Blass
J P
JP
Cooper
A J
AJ
D000818:Animals; D002103:Cadaverine / Q000031:analogs & derivatives / Q000378:metabolism; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors / Q000032:analysis*; D005453:Fluorescence; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D006168:Guinea Pigs; D006657:Histones / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011107:Polylysine / Q000378:metabolism; D011485:Protein Binding; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D001615:beta-Endorphin / Q000378:metabolism
D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D006657:Histones; D011107:Polylysine; D001615:beta-Endorphin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; C008542:monodansylcadaverine; D002103:Cadaverine