ANALYTICAL BIOCHEMISTRY 70, 9 4 - 9 9 (1976)
Permeabilization of Yeast for Enzyme Assays: An Extremely Simple Method for Small Samples DEBORAH BERNHARDT
MOWSHOWITZ
Department of Biological Sciences, Columbia University, Broadway and ll6th Street, New York, New York 10027 Received J u n e 19, 1975; accepted A u g u s t 8, 1975 It has been found that c~-glucosidase can be a s s a y e d quantitatively in small samples of u n w a s h e d yeast cells permeabilized by drying on filters at r o o m temperature. T h e details of the procedure are discussed together with the possible application of the permeabilization m e t h o d to the quantitative a s s a y of other e n z y m e s and to the qualitative a s s a y of c~-glucosidase and other e n z y m e s in cells grown on solid media.
Yeast cells possess an extremely tough cell wall that makes quantitative breakage of multiple cell samples extremely difficult. The effort involved in processing multiple samples for enzyme assays can be reduced considerably by breaking many small samples at once by using a multiple sample holder, recently described for the Braun homogenizer (1), or by permeabilizing the yeast cells with D M S O 1 (2) or toluene (3), or by freeze drying (4) so that breakage is avoided entirely. A new permeabilization method is described below that utilizes the permeabilizing effects of drying. It does not require any special equipment, uses very small samples (20-100 /zl), and is much easier to carry out than any of the methods referred to above, because it does not involve any washing or centrifuging of the cells. MATERIALS AND METHODS
Strains and Growth of Cells Many different haploid and diploid strains ofSaccharomyces cerevisiae and S. carlsbergensis and hybrids between them have been used in these experiments. Reproducible results have been obtained with all strains tested but the relative efficiencies of permeabilization of the different strains have not been checked. The constitutive strain used for the experiment of Fig. 3 was derived from a strain ofS. carlsbergensis obtained from R. Needleman. In this strain o~-glucosidase production is repressed by glucose but not affected by maltose. The yeast were grown in either YP 1 Abbreviations: D M S O , dimethyl sulfoxide; P N P G , p-nitrophenyl-~-D-glucoside. 94 Copyright © 1976by AcademicPress, lnc All rights of reproduction in any form reserved
P E R M E A B I L I Z A T I O N OF YEAST
95
broth (1% yeast extract, 2% peptone, 2% carbon source) or the semidefined medium of Van Wijk (5) which contains no peptone and a low concentration of yeast extract. The liquid cultures were grown in a shaking water-bath or on a roller apparatus in regular Pyrex test tubes that fit into a standard Klett-Summerson colorimeter. Growth of the cultures was monitored by following the absorbance in a Klett-Summerson colorimeter equipped with a red filter.
Sampling and Permeabilization Samples for enzyme as says (10-100 ~1) were withdrawn with disposable glass micropipets or with samplers fitted with disposable plastic tips. Samples were applied to 23-mm disks of Whatman 3 MM filter paper (scintillation pads) which had been numbered in pencil. It was found that the samples must be applied in a consistent manner (always as one spot or always spread out, etc.) to get reproducible results. The exact amount of enzyme activity detected varies with the spreading technique but is highly reproducible for any one method. The filters were then dried at room temperature for a convenient length of time, usually overnight. It was found that a-glucosidase activity reaches a maximum after 2-3 hr of drying at room temperature or in a few minutes of drying with a hair dryer used at cold temperature; it remains constant for at least a week (data not shown).
Enzyme Assays Activity ofc~-glucosidase was determined by measuring the hydrolysis of P N P G at 410 nm (6). The dried filters were placed in glass scintillation vials, and 1 ml of a 3.33 mM P N P G solution (1 mg/ml in 0.05 M potassium phosphate buffer (pH 6.8)) was added to each vial. Mercaptoethanol or dithiothreitol was added to the P N P G solution to a concentration of 10-~ M in some experiments. The vials were then capped and placed in a shaking H20-bath at 30°C for 2-30 rain. To stop the reaction the vials were placed in a 95°C-H20 bath for 2 min. Controls (not shown) indicated that it takes ½ min to stop the reaction under these conditions so incubation times were recorded as the time incubated at 30°C + ½ rain. The samples were cooled to room temperature and diluted with phosphate buffer if necessary, and the absorbance was determined in a Gilford or Zeiss spectrophotometer at 410 nm. The a-glucosidase activity is expressed as absorbance measured × dilution.
Preparation of Extracts Cells in growth medium were centrifuged, washed once with distilled H20, and resuspended in 0.05 M potassium phosphate buffer (pH 7.4)
96
DEBORAH BERNHARDT MOWSHOWITZ
containing 10-3 M mercaptoethanol. The suspensions were mixed with an approximately equal volume of 0.45-mm glass beads and homogenized for two 1-min intervals in a Braun cell homogenizer using the holder described by Needleman and Tzagoloff (1). The homogenate containing the glass beads was centrifuged at low speed, and the supernatant fluid was removed. Fifty-microliter portions of the supernatant solution were placed in scintillation vials and assayed in exactly the same manner as the permeabilized cell samples.
Results and Discussion In an attempt to modify the DMSO permeabilization method of Adams (2), yeast cells were applied to filters and the filters were processed in batch (washed in HzO, incubated in DMSO, and washed extensively in H20). It was discovered by accident that drying on filters was sufficient to permeabilize the cells for assay of ct-glucosidase and that additional manipulations were not necessary. When dried cells were assayed at increasing concentrations of PN PG to determine the optimal substrate concentration for routine assays, it was found that the apparent Km of a-glucosidase for P N P G in dried cells is about 2 mM (data not shown). This is much higher than the values of about 0.2 mM reported by others for a-glucosidase in extracts (5), in cells permeabilized with DMSO (2), or in extracts of the same cells in this
?
INCUBATION TIME
B
I ~
41 81 ~CELLS/F[LIER (X6XlO 6)
116
FIG. 1. Dependence of color production on incubation time and number of cells per filter. A culture grown in YP maltose containing approximately 5 x 107 cells/ml was diluted 1:2, 1:4, 1:8, and 1:16 with YP maltose, and 50-/xl samples of each dilution were applied to filters, dried, and incubated with P N P G for appropriate periods of time as described under Materials and Methods. (A) Color production vs. incubation time with different dilutions of the culture, Dilutions: (A), none; (~), 1:2; (O), 1:4; (11), 1:8; (O), 1:16. (B) Color production vs number of cells per filter at different incubation times: (A), 15.5 min; (~), 12.5 min; (O), 9.5 min; (11), 6.5 min; and (Q), 3.5 min.
P E R M E A B I L I Z A T I O N OF YEAST
97
"o
z
Permeabilization of Yeast for Enzyme Assays: An Extremely Simple Method for Small Samples DEBORAH BERNHARDT
MOWSHOWITZ
Department of Biological Sciences, Columbia University, Broadway and ll6th Street, New York, New York 10027 Received J u n e 19, 1975; accepted A u g u s t 8, 1975 It has been found that c~-glucosidase can be a s s a y e d quantitatively in small samples of u n w a s h e d yeast cells permeabilized by drying on filters at r o o m temperature. T h e details of the procedure are discussed together with the possible application of the permeabilization m e t h o d to the quantitative a s s a y of other e n z y m e s and to the qualitative a s s a y of c~-glucosidase and other e n z y m e s in cells grown on solid media.
Yeast cells possess an extremely tough cell wall that makes quantitative breakage of multiple cell samples extremely difficult. The effort involved in processing multiple samples for enzyme assays can be reduced considerably by breaking many small samples at once by using a multiple sample holder, recently described for the Braun homogenizer (1), or by permeabilizing the yeast cells with D M S O 1 (2) or toluene (3), or by freeze drying (4) so that breakage is avoided entirely. A new permeabilization method is described below that utilizes the permeabilizing effects of drying. It does not require any special equipment, uses very small samples (20-100 /zl), and is much easier to carry out than any of the methods referred to above, because it does not involve any washing or centrifuging of the cells. MATERIALS AND METHODS
Strains and Growth of Cells Many different haploid and diploid strains ofSaccharomyces cerevisiae and S. carlsbergensis and hybrids between them have been used in these experiments. Reproducible results have been obtained with all strains tested but the relative efficiencies of permeabilization of the different strains have not been checked. The constitutive strain used for the experiment of Fig. 3 was derived from a strain ofS. carlsbergensis obtained from R. Needleman. In this strain o~-glucosidase production is repressed by glucose but not affected by maltose. The yeast were grown in either YP 1 Abbreviations: D M S O , dimethyl sulfoxide; P N P G , p-nitrophenyl-~-D-glucoside. 94 Copyright © 1976by AcademicPress, lnc All rights of reproduction in any form reserved
P E R M E A B I L I Z A T I O N OF YEAST
95
broth (1% yeast extract, 2% peptone, 2% carbon source) or the semidefined medium of Van Wijk (5) which contains no peptone and a low concentration of yeast extract. The liquid cultures were grown in a shaking water-bath or on a roller apparatus in regular Pyrex test tubes that fit into a standard Klett-Summerson colorimeter. Growth of the cultures was monitored by following the absorbance in a Klett-Summerson colorimeter equipped with a red filter.
Sampling and Permeabilization Samples for enzyme as says (10-100 ~1) were withdrawn with disposable glass micropipets or with samplers fitted with disposable plastic tips. Samples were applied to 23-mm disks of Whatman 3 MM filter paper (scintillation pads) which had been numbered in pencil. It was found that the samples must be applied in a consistent manner (always as one spot or always spread out, etc.) to get reproducible results. The exact amount of enzyme activity detected varies with the spreading technique but is highly reproducible for any one method. The filters were then dried at room temperature for a convenient length of time, usually overnight. It was found that a-glucosidase activity reaches a maximum after 2-3 hr of drying at room temperature or in a few minutes of drying with a hair dryer used at cold temperature; it remains constant for at least a week (data not shown).
Enzyme Assays Activity ofc~-glucosidase was determined by measuring the hydrolysis of P N P G at 410 nm (6). The dried filters were placed in glass scintillation vials, and 1 ml of a 3.33 mM P N P G solution (1 mg/ml in 0.05 M potassium phosphate buffer (pH 6.8)) was added to each vial. Mercaptoethanol or dithiothreitol was added to the P N P G solution to a concentration of 10-~ M in some experiments. The vials were then capped and placed in a shaking H20-bath at 30°C for 2-30 rain. To stop the reaction the vials were placed in a 95°C-H20 bath for 2 min. Controls (not shown) indicated that it takes ½ min to stop the reaction under these conditions so incubation times were recorded as the time incubated at 30°C + ½ rain. The samples were cooled to room temperature and diluted with phosphate buffer if necessary, and the absorbance was determined in a Gilford or Zeiss spectrophotometer at 410 nm. The a-glucosidase activity is expressed as absorbance measured × dilution.
Preparation of Extracts Cells in growth medium were centrifuged, washed once with distilled H20, and resuspended in 0.05 M potassium phosphate buffer (pH 7.4)
96
DEBORAH BERNHARDT MOWSHOWITZ
containing 10-3 M mercaptoethanol. The suspensions were mixed with an approximately equal volume of 0.45-mm glass beads and homogenized for two 1-min intervals in a Braun cell homogenizer using the holder described by Needleman and Tzagoloff (1). The homogenate containing the glass beads was centrifuged at low speed, and the supernatant fluid was removed. Fifty-microliter portions of the supernatant solution were placed in scintillation vials and assayed in exactly the same manner as the permeabilized cell samples.
Results and Discussion In an attempt to modify the DMSO permeabilization method of Adams (2), yeast cells were applied to filters and the filters were processed in batch (washed in HzO, incubated in DMSO, and washed extensively in H20). It was discovered by accident that drying on filters was sufficient to permeabilize the cells for assay of ct-glucosidase and that additional manipulations were not necessary. When dried cells were assayed at increasing concentrations of PN PG to determine the optimal substrate concentration for routine assays, it was found that the apparent Km of a-glucosidase for P N P G in dried cells is about 2 mM (data not shown). This is much higher than the values of about 0.2 mM reported by others for a-glucosidase in extracts (5), in cells permeabilized with DMSO (2), or in extracts of the same cells in this
?
INCUBATION TIME
B
I ~
41 81 ~CELLS/F[LIER (X6XlO 6)
116
FIG. 1. Dependence of color production on incubation time and number of cells per filter. A culture grown in YP maltose containing approximately 5 x 107 cells/ml was diluted 1:2, 1:4, 1:8, and 1:16 with YP maltose, and 50-/xl samples of each dilution were applied to filters, dried, and incubated with P N P G for appropriate periods of time as described under Materials and Methods. (A) Color production vs. incubation time with different dilutions of the culture, Dilutions: (A), none; (~), 1:2; (O), 1:4; (11), 1:8; (O), 1:16. (B) Color production vs number of cells per filter at different incubation times: (A), 15.5 min; (~), 12.5 min; (O), 9.5 min; (11), 6.5 min; and (Q), 3.5 min.
P E R M E A B I L I Z A T I O N OF YEAST
97
"o
z
