Localization of the cholesterol oxidase in Rhodococcus erythropolis IMET 7185 studied by immunoelectron microscopy

Rhodococcus erythropolis IMET 7185 produces an inducible cholesterol oxidase (COD) which can easily be extracted by treatment of cells with 0.1% Triton X-100. The yield of the enzyme was 3.3 U/g wet wt from induced cells, which is about 5 times more than from non-induced cells. A study of the location of COD on intact cells and on ultrathin sections by means of immunogold electron microscopy revealed the following distribution, which corresponds with the biochemical results: (1) COD, which is extractable by the detergent, was localized in a distance up to 80 nm above the cell surface. It belongs to a surface layer, which only became visible after lysine/glutaraldehyde treatment and staining with ruthenium red, indicating a high carbohydrate content. (2) Non-extractable COD was found on the cell surface in a shorter distance to the cell wall as well as within the cell wall, in the cytoplasmic membrane and in the peripheral cytoplasm. In the latter clusters of gold particles on some places suggest the presence of larger amounts of insoluble enzyme.