SUMMARY: Conditions are described for the preparation of permeabilized cells of This method has been used for the assay of enzymes in both yeast cells and germ-tube forming cells. A mixture of toluene/ethanol/Triton X-100 (1:4:0.2, by vol.) at 15% (v/v) and 8% (v/v) was optimal for the assay of glucose-6-phosphate dehydrogenase in yeast and germ-tube forming cells, respectively. The concentration of toluene/ethanol/Triton X-100 required for optimal activity of other enzymes was influenced by the cellular location of the enzyme, growth phase and morphology. The membrane-bound enzymes (chitin synthase, glucan synthase, ATPase), cytosolic enzymes (glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, pyruvate kinase, phosphofructokinase, alkaline phosphatase, glucosamine-6-phosphate deaminase and -acetylglucosamine kinase) and wall enzymes (-glucosidase and acid phosphatase) were measured and compared to the activity obtained in cell extracts. The pattern of enzyme induction and the properties of the allosteric enzymes phosphofructokinase and pyruvate kinase were measured Pyruvate kinase was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a of 1.0 mM. The for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. phosphofructokinase was homotropic for fructose 6-phosphate ( of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured or were similar for both yeast cells and germ-tube forming cells.


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