| Autor: |
Loewy, Ariel G., Blodgett, James K., Blase, Frances R., May, Matthew H. |
| Zdroj: |
Analytical Biochemistry; March 1997, Vol. 246 Issue: 1 p111-117, 7p |
| Abstrakt: |
We have developed a substrate to assay for an isopeptidase, an enzyme capable of cleaving theNϵ-(γ-glutamic) lysine bond which crosslinks polypeptide chains. This substrate consists of modified lysine (N-α-[3H]acetyl-l-lysine-N-methylamide or ALMA), linked by its ϵ-amino group to a γ-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 ofBacillus cereusand 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 10−8μmol of [3H]ALMA/min/μg 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 γ-glutamyl transpeptidase, γ-glutamyl cyclotransferase, and the reversal of transglutaminase. We conclude that an isopeptidase mechanism most plausibly accounts for the ALMA release. |
| Databáze: |
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