Yeast cell permeabilization by osmotic shock allows determination of enzymatic activities in situ.
Yeast cells were permeabilized by incubation in 0.8 M sorbitol followed by suspension in dilute buffer. A preincubation with 2-mercaptoethanol was also included for optimal permeabilization. More than 90% of the treated cells were stainable with methylene blue. Determinations of cell wall-synthesizing enzymes (beta(1 --> 3)glucan and chitin synthases) and cytosolic enzymes in permeabilized cells yielded similar or higher activities than those in cell extracts. With chitin synthase III, the activity obtained with cells was 4- to 6-fold higher than in membrane preparations. Little protein leaks from the cells during permeabilization; yet the cells appear to be readily permeable to substrates and even proteins. Thus, these preparations may be of wide use for the study of enzymes and of biological processes in situ.
Crotti
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LB
National Institute of Diabetes and Digestive and Kidney Diseases, Laboratory of Biochemistry and Genetics, Bethesda, Maryland 20892, USA.
Drgon
T
T
Cabib
E
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D002463:Cell Membrane Permeability / Q000502:physiology; D002687:Chitin Synthase / Q000032:analysis*; D005964:Glucosyltransferases / Q000032:analysis*; D006003:Glycogen / Q000096:biosynthesis; D009997:Osmotic Pressure; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D013012:Sorbitol / Q000494:pharmacology; D000516:alpha-Amylases / Q000378:metabolism
D013012:Sorbitol; D006003:Glycogen; D005964:Glucosyltransferases; C040232:glucan synthase; D002687:Chitin Synthase; D000516:alpha-Amylases