Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy.
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.
D002458:Cell Fractionation / Q000379:methods; D002500:Centrifugation, Isopycnic; D004798:Enzymes / Q000494:pharmacology; D015151:Immunoblotting; D007472:Iohexol; D008830:Microbodies / Q000648:ultrastructure*; D016253:Microscopy, Immunoelectron; D010408:Penicillium chrysogenum / Q000648:ultrastructure*; D011523:Protoplasts / Q000648:ultrastructure
D004798:Enzymes; C045154:novozym 234; D007472:Iohexol