Isolation and partial characterization of a plasma membrane-enriched fraction of Blastomyces dermatitidis

The isolation and characterization of a plasma membrane-enriched fraction is reported from the pathogenic fungus Blastomyces dermatitidis employing a simplified and inexpensive procedure. The yeast cells, suspended in an osmotic stabilizer buffer, were subjected to gentle mechanical disruption followed by a three-step differential centrifugation to obtain a 40,000 x g sediment. This fraction showed 2.7- and 3.3-fold relative enrichment of the plasma membrane marker enzymes Mg2(+)-ATPase and 5'-nucleotidase, respectively, with modest cross-contamination of other intracellular membranes. The fraction comprised approximately 49% protein, 31% carbohydrate, 11% phospholipids and 9% sterol with sterol to phospholipid molar ratio of 1.1. Transmission electron microscopy of the plasma membrane-enriched fraction showed homogeneous vesicles with a tripartite unit membrane of 115 A thickness.