A sensitive fluorometric assay for tissue transglutaminase

Anal Biochem. 2001 May 15;292(2):198-206. doi: 10.1006/abio.2001.5040.

Abstract

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).

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Cadaverine / analogs & derivatives
  • Cadaverine / metabolism
  • Cross-Linking Reagents
  • Enzyme Inhibitors / analysis*
  • Fluorescence
  • GTP-Binding Proteins / antagonists & inhibitors*
  • GTP-Binding Proteins / metabolism*
  • Guinea Pigs
  • Histones / metabolism
  • Kinetics
  • Liver / enzymology*
  • Polylysine / metabolism
  • Protein Binding
  • Protein Glutamine gamma Glutamyltransferase 2
  • Transglutaminases / antagonists & inhibitors*
  • Transglutaminases / metabolism*
  • beta-Endorphin / metabolism

Substances

  • Cross-Linking Reagents
  • Enzyme Inhibitors
  • Histones
  • Polylysine
  • beta-Endorphin
  • Protein Glutamine gamma Glutamyltransferase 2
  • Transglutaminases
  • GTP-Binding Proteins
  • monodansylcadaverine
  • Cadaverine