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

Analytical Biochemistry
S Sesták, V Farkas

Abstract

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.

References

Aug 1, 1976·Cryobiology·F Dankberg, M D Persidsky
Jan 1, 1973·Experimental Cell Research·B L Carter, H O Halvorson
Sep 1, 1971·Proceedings of the National Academy of Sciences of the United States of America·E Cabib, V Farkas
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May 24, 1994·Proceedings of the National Academy of Sciences of the United States of America·W J ChoiE Cabib

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Citations

Mar 13, 2009·The Journal of Biological Chemistry·Vishukumar AimaniandaJean-Paul Latgé
Apr 17, 2008·Journal of Industrial Microbiology & Biotechnology·Jessy Abraham, S G Bhat
Apr 3, 2004·Biotechnology Progress·Chi-Kin Chow, Sean P Palecek
Feb 7, 2009·Letters in Applied Microbiology·F VillaC Sorlini
Mar 13, 2009·Environmental Microbiology·Thomas D NiederbergerLyle G Whyte
Jan 8, 2017·Journal of Animal Physiology and Animal Nutrition·J B LiuH F Zhang
Aug 14, 2020·Journal of the Science of Food and Agriculture·Xinlei ShenYuying Shuai

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