The in situ Assay of Candida albicans Enzymes during Yeast Growth and Germ-tube Formation

Abstract

Conditions are described for the preparation of permeabilized cells of Candida albicans. This method has been used for the in situ 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 in situ 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 in situ 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 N-acetylglucosamine kinase) and wall enzymes (beta-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 in situ. Pyruvate kinase in situ was homotropic for phosphoenolpyruvate with a Hill coefficient of 1.9 and a S0.5 of 0.6 mM, whereas in cell extracts, it had a Hill coefficient of 1.9 and a S0.5 of 1.0 mM. The Km for ATP was 1.6 mM in cell extracts and 1.8 mM in permeabilized cells. In situ phosphofructokinase was homotropic for fructose 6-phosphate (S0.5 of 2.3 mM, Hill coefficient of 4.0). The kinetic properties of pyruvate kinase and phosphofructokinase measured in situ or in vitro were similar for both yeast cells and germ-tube forming cells.

Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation

“…1 . 58), pglucosidase (EC 3.2.1.21) and acid phosphatase (EC 3.1 .3.2) were assayed as described by Ram et al (1983Ram et al ( , 1984. aGlucosidase (EC 3.2.1 .20, pH 7*0), P-galactosidase (EC 3 .…”

The Secretion of N-Acetylglucosaminidase during Germ-tube Formation in Candida albicans

“…Cell-free extracts were prepared in either 0.01 M-or 0.1 M-potassium phosphate buffer, pH 6-95, as indicated, with a Braun homogenizer (B. Braun, Melsungen, FRG) (Gopal et al, 1982). Permeabilized cells were prepared for in situ assays essentially as described by Ram et al (1983); cell suspensions (1.2-1.6 x lo9 cells ml-' in 0.1 M-potassium phosphate buffer, containing 0.01 M-M~CI,) were shaken with 10% (v/v) toluene'ethanoliTriton X-100 ( 1 :4 :0.2, by vol.) for 5 min at 4 "C and washed twice with 0.1 M-potassium phosphate buffer, pH 6.95.…”

The Cellular Location of Proteases in Candida albicans

“…1 .42) and glucose-6-phosphate dehydrogenase (EC 1.1 . 1.49) were assayed as described by Ram et al (1983) except that for glucose-6-phosphate dehydrogenase each assay also contained 50 pmol glucose 6-phosphate and 0.3 pmol NADP+. Succinate dehydrogenase (EC 1 .3.99.1) was assayed as described by Hubbard et al (1986).…”