Synthesis and use of a substrate for the detection of isopeptidase activity

We have developed a substrate to assay for an isopeptidase, an enzyme capable of cleaving the Nepsilon-(gamma-glutamic) lysine bond which crosslinks polypeptide chains. This substrate consists of modified lysine (N-alpha-[3H]acetyl-l-lysine-N-methylamide or ALMA), linked by its epsilon-amino group to a gamma-carboxyl amide group of casein with guinea pig liver transglutaminase or Factor XIIIa. We used this substrate to demonstrate the release of [3H]ALMA from [3H]ALMA-casein in a culture medium of Bacillus cereus and in brain homogenates of 12- to 14-day-old embryonic chicks. The prokaryotic and the eukaryotic enzymes resemble each other in that both are activated by Ca2+ or Mg2+ and by alkaline phosphatase and both are inhibited by ATP. The [3H]ALMA-casein is a sensitive substrate able to measure reliably specific activities as low as 10(-8) micromol of [3H]ALMA/min/microg protein. The special advantage of this substrate is that the initial rate of ALMA-casein cleavage is not affected significantly by the levels of protease contaminants we have encountered. We were able to rule out alternative mechanisms such as gamma-glutamyl transpeptidase, gamma-glutamyl cyclotransferase, and the reversal of transglutaminase. We conclude that an isopeptidase mechanism most plausibly accounts for the ALMA release.