A spheroplast rate assay for determination of cell wall integrity in yeast.
The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1-5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5-15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls.
Ovalle
R
R
Department of Biological Sciences, Hunter College, City University of New York, New York 10021, USA.
Lim
S T
ST
Holder
B
B
Jue
C K
CK
Moore
C W
CW
Lipke
P N
PN
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology; D002176:Candida albicans / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D002473:Cell Wall / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D005296:Ferrous Compounds / Q000494:pharmacology; D005656:Fungal Proteins / Q000235:genetics; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000502:physiology / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry; D013104:Spheroplasts / Q000737:chemistry / Q000254:growth & development*; D013997:Time Factors
D000900:Anti-Bacterial Agents; D005296:Ferrous Compounds; D005656:Fungal Proteins; C064013:KRE1 protein, S cerevisiae; D008562:Membrane Glycoproteins; D000644:Quaternary Ammonium Compounds; D029701:Saccharomyces cerevisiae Proteins; D001761:Bleomycin; C037833:zymolyase; C038178:ammonium ferrous sulfate; D006867:Hydrolases