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 N-epsilon-(gamma-glutamic) lysine bond which c rosslinks polypeptide chains, This substrate consists of modified lysi ne (N-alpha-[H-3]acetyl-L-lysine-N-methylamide or ALMA), linked by its E-amino group to a gamma-carboxyl amide group of casein with guinea p ig liver transglutaminase or Factor XIIIa, We used this substrate to d emonstrate the release of [H-3]ALMA from [H-3]ALMA-casein in a culture medium of Bacillus cereus and in brain homogenates of 12- to 14-day-o ld embryonic chicks, The prokaryotic and the eukaryotic enzymes resemb le each other in that both are activated by Ca2+ or Mg2+ and by alkali ne phosphatase and both are inhibited by ATP, The [H-3]ALMA-casein is a sensitive substrate able to measure reliably specific activities as low as 10(-8) mu mol of [H-3]ALMA/min/mu g protein. The special advant age 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 s uch as gamma-glutamyl transpeptidase, gamma-glutamyl cyclotransferase, and the reversal of transglutaminase. We conclude that an isopeptidas e mechanism most plausibly accounts for the ALMA release. (C) 1997 Aca demic Press.