Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.

Détails

ID Serval
serval:BIB_89E90D35B10D
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.
Périodique
Biochimica et Biophysica Acta - General Subject
Auteur(s)
Müller W.H., Essers J., Humbel B.M., Verkleij A.J.
ISSN
0304-4165
ISSN-L
1872-8006
Statut éditorial
Publié
Date de publication
1995
Volume
1245
Numéro
2
Pages
215-220
Langue
anglais
Résumé
A procedure to enrich microbodies from Penicillium chrysogenum and a method to evaluate the purity and integrity of the microbodies are described. As a P. chrysogenum microbody marker acyltransferase (AT) was used. The P. chrysogenum hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from mycelial debris after 10,000 x g centrifugation. Purified protoplasts were lysed, and the cell homogenate was centrifuged to form a 14,000 x g pellet. After 2 h, 45,000 x g isopycnic centrifugation of the 14,000 x g pellet on a continuous 20-60% nycodenz gradient, ten fractions were collected. The fractions were analyzed for AT containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that AT-microbodies are enriched in the 38% nycodenz fraction. The microbodies had a diameter of 400 to 500 nm, revealed an intact single membrane and confined AT. The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.
Mots-clé
Cell Fractionation/methods, Centrifugation, Isopycnic, Enzymes/pharmacology, Immunoblotting, Iohexol, Microbodies/ultrastructure, Microscopy, Immunoelectron, Penicillium chrysogenum/ultrastructure, Protoplasts/ultrastructure
Pubmed
Web of science
Création de la notice
18/10/2012 13:55
Dernière modification de la notice
20/08/2019 14:48
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