Yeast cell permeabilization by osmotic shock allows determination of enzymatic activities in situ

Analytical Biochemistry
L B CrottiE Cabib

Abstract

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.

References

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Citations

Jul 28, 2013·Antonie van Leeuwenhoek·Joanna BerlowskaWojciech Ambroziak
Aug 13, 2008·The Journal of Biological Chemistry·Enrico CabibPeter McPhie
Mar 13, 2009·The Journal of Biological Chemistry·Vishukumar AimaniandaJean-Paul Latgé
Aug 16, 2002·Molecular Biology of the Cell·Dong-Hyun RohEnrico Cabib
Jun 13, 2003·Molecular Biology of the Cell·Martin SchmidtEnrico Cabib
Aug 17, 2011·Antimicrobial Agents and Chemotherapy·Scott S WalkerTodd A Black
Feb 6, 2007·Applied and Environmental Microbiology·Silke HagenVera Meyer
May 23, 2002·Analytical Biochemistry·Héctor A LuceroDorota A Bulik
Jan 15, 2011·International Journal of Microbiology·Martin SchmidtMichael P Boyer
Dec 2, 2008·European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences·I PieperM Bertau
Apr 3, 2004·Biotechnology Progress·Chi-Kin Chow, Sean P Palecek
Jun 17, 2003·The Journal of Biological Chemistry·Xiaotian ZhongGuido Guidotti
Oct 19, 2012·Journal of Cell Science·Noelia BlancoEnrico Cabib
Aug 2, 2001·Yeast

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