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.
Loewy
A G
AG
Department of Biology, Haverford College, Haverford, Pennsylvania, 19041, USA.
Blodgett
J K
JK
Blase
F R
FR
May
M H
MH
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D019759:Carbon-Nitrogen Lyases; D002364:Caseins / Q000378:metabolism; D002642:Chick Embryo; D002855:Chromatography, Thin Layer; D006168:Guinea Pigs; D008099:Liver / Q000201:enzymology; D008190:Lyases / Q000378:metabolism*; D008239:Lysine / Q000031:analogs & derivatives*; D008274:Magnesium / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism
D002364:Caseins; C104976:N(alpha)-acetyllysine-N-methylamide; D011503:Transglutaminases; D008190:Lyases; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D008274:Magnesium; D008239:Lysine; D002118:Calcium