N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain
Détails
ID Serval
serval:BIB_4C198FFAFD48
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
N-acetylmuramoyl-L-alanine amidase assay based on specific radioactive labeling of muropeptide L-alanine: quantitation of the enzyme activity in the autolysin deficient Bacillus subtilis 168, flaD strain
Périodique
Anal Biochem
ISSN
0003-2697 (Print)
Statut éditorial
Publié
Date de publication
10/1991
Volume
198
Numéro
1
Pages
15-8
Notes
Comparative Study
Journal Article --- Old month value: Oct
Journal Article --- Old month value: Oct
Résumé
A sensitive and highly reproducible assay for N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28) was devised, based on specific and homogeneous L-[14C]alanine labeling of the substrate, the peptidoglycan. The method involves partial purification of both the enzyme and the substrate and monitoring the muropeptide cleavage by coupling fluorodinitrobenzene to freed L-alanine NH2 groups. After acid hydrolysis of the substrate, the resulting DNP-L-alanine and L-alanine are separated by TLC, and radioactive counts in relevant spots are determined. Application of the method to the autolysin-endowed strain and an autolysis-deficient flaD-bearing mutant has revealed (i) that the N-acetylmuramoyl-L-alanine amidase behaves like an endoenzyme with an apparent Kcat(s-1) of 40, and (ii) that the residual enzyme activity in the flaD bearing strain amounts to 2.5 (+/- 0.1)% of that of the parent strain.
Mots-clé
Alanine/metabolism
Amines/analysis
Bacillus subtilis/*enzymology
Cell Wall/*enzymology
Chromatography, Thin Layer
Dinitrofluorobenzene/chemistry
N-Acetylmuramoyl-L-alanine Amidase/*analysis/metabolism
Peptidoglycan/*metabolism
Phenylalanine/analogs & derivatives/analysis
Pubmed
Web of science
Création de la notice
24/01/2008 15:07
Dernière modification de la notice
20/08/2019 15:00