In situ assays of fungal enzymes in cells permeabilized by osmotic shock.
Permeabilization of yeast and other fungal cells by osmotic shock enabled the in situ assays of intracellular plasma membrane-bound enzymes, such as beta-1,3-glucan synthase, chitin synthase, and Na(+)/K(+) ATPase as well as the soluble, cytoplasmic enzymes, such as lactate dehydrogenase and alpha-glucosidase. The permeabilization was accomplished by rapid changes in osmolarity of the washing buffer at 0 degrees C whereby 0.5-3.5 M glycerol, sorbitol, and/or mannitol and/or 1 M KCl could be used as the osmolytes. No appreciable leakage of intracellular proteins occurred during the permeabilization procedure. The described procedure caused practically complete cell permeabilization while avoiding treatments with organic solvents, detergents, and other xenobiotics currently used for the permeabilization of microbial cells.
Sesták
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Institute of Chemistry, Slovak Academy of Sciences, Dúbravská cesta 9, Bratislava, 84238, Slovakia.
Farkas
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D002687:Chitin Synthase / Q000032:analysis*; D005964:Glucosyltransferases / Q000032:analysis*; D009997:Osmotic Pressure; D010539:Permeability; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism
D005964:Glucosyltransferases; C040232:glucan synthase; D002687:Chitin Synthase