High-throughput scintillation proximity assay for transglutaminase activity measurement.
Members of the transglutaminase enzyme family are involved in a broad range of biological phenomena, including haemostasis, apoptosis, semen coagulation, skin formation, and wound healing. A new and rapid method for measurement of transglutaminase activity is described in this article. The enzyme links tritium-labeled putrescine to biotinylated oligoglutamine, and the tritiated peptide is bound to a streptavidin-coated microtiter plate permanently covered by a thin layer of scintillant. Only the radioisotope incorporated into the peptide substrate is close enough to the scintillant molecules for photons to be produced. The signal generation depends on the transglutaminase activity, and it can be detected by appropriate light-measuring instrumentation without separation steps. The assay is sensitive, specific, linear at concentrations of tissue transglutaminase between 0.05 and 1.6m U/ml, and suitable for high-throughput measurements.
Research Center for Molecular Medicine, Medical and Health Science Center, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98, Hungary.
András
A
Mádi
Levente
L
Kárpáti
András
A
Kovács
László
L
Muszbek
László
L
Fésüs
D000818:Animals; D001710:Biotin / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D007700:Kinetics; D051379:Mice; D010455:Peptides / Q000737:chemistry; D011700:Putrescine / Q000494:pharmacology; D012107:Research Design; D012588:Scintillation Counting / Q000379:methods*; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D001710:Biotin; D006160:Guanosine Triphosphate; D011503:Transglutaminases; D002118:Calcium; D011700:Putrescine