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 als o included for optimal permeabilization, More than 90% of the treated cells were stainable with methylene blue, Determinations of cell wall-synthesizi ng enzymes (beta (1 --> 3)glucan and chitin synthases) and cytosolic enzyme s 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; Set 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, (C) 2001 Academic Press.