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

@entity20124 @entity5449 @entity5922
A procedure to enrich microbodies from @entity5449 and a method to evaluate the purity and integrity of the microbodies are described. As a @entity5449 microbody marker @entity20124 ( @entity20124 ) was used. The @entity5449 hyphae were converted into protoplasts with Novozym 234. In Percoll-sucrose buffer the protoplasts were separated from @entity5922 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 @entity20124 containing microbodies by immuno-blotting and immuno-electron microscopy. The results showed that @entity20124 -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 @entity20124 . The estimated equilibrium density of the P. chyrsogenum microbodies was 1.20 g ml-1 as deduced from the 38% (w/v) nycodenz concentration.
