In situ assays of fungal enzymes in cells permeabilized by osmotic shock

Permeabilization of yeast and other fungal cells by osmotic shock enabled t he in situ assays of intracellular plasma membrane-bound enzymes, such as b eta -1,3-glucan synthase, chitin synthase, and Na+/R+ ATPase as well as the soluble, cytoplasmic enzymes, such as lactate dehydrogenase and cu-glucosi dase. The permeabilization was accomplished by rapid changes in osmolarity of the washing buffer at 0 degreesC whereby 0.5-3.5 M glycerol, sorbitol, a nd/or mannitol and/or I M KCI could be used as the osmolytes, No appreciabl e leakage of intracellular proteins occurred during the permeabilization pr ocedure. The described procedure caused practically complete cell permeabil ization while avoiding treatments with organic solvents, detergents, and ot her xenobiotics currently used for the permeabilization of microbial cells. (C) 2001 Academic Press.