ANALYTICAL
BIOCHEMISTRY
A Simple
96,
152-154
Whole-Ceil
(1979)
Assay for E. co/i Anthranilate
Synthetase
RICHARD D. BLISS Department
of Biology,
University Received
of Cai$ornia, September
Copytight 0 1979 by Academic Press. Inc. All tights of reproduction in my form reserved.
California
92521
29, 1978
used successfully to decryptify the same enzyme in yeast (4). The method reported here combines decryptification and batch assay of the E. coii enzyme in a single step. The assay is a modification of the technique of Egan and Gibson (2), who used ethyl acetate to extract the product anthranihc acid from an acid-stopped reaction mixture. I have found that the ethyl acetate may also be used to decryptify the enzyme in whole cells, and, thus, the same reagent may be used to perform two functions.
Anthranilate synthetase, the first enzyme of the tryptophan biosynthetic pathway, is usually assayed in cell-free extracts by determining the rate of appearance of the fluorescent product, anthranilic acid (1,2). The reaction is usually carried out in a spectrofluorometer cuvette while it is in the instrument, and the increase in product fluorescence is recorded on a strip chart. While this assay has proved to be useful in studies of the enzyme’s properties, it is cumbersome for derepression experiments and other studies in which intracellular enzyme specific activity is the parameter of interest. Since the assays must be carried out sequentially, substantial amounts of time may be involved in preparing reaction mixtures, rinsing cuvettes, and recording the data. Furthermore, preparation of a large number of cell-free extracts, either by sonication or other physical means, adds considerably to the labor per assay. Specific activity measurements may be simplified by the use of stopped assays, which permit the reaction to be carried out for several samples simultaneously. A further simplification is possible through the use of a whole-cell assay, in which the cells are treated chemically to decryptify the enzyme activity, thus avoiding the necessity for physical disruption. Suitable methods of decryptification vary with the enzyme activity under investigation, but treatment with a mixture of lysozyme, detergent, and ethylenediaminetetraacetate (EDTA) has been reported as being satisfactory for use with Escherichia coli anthranilate synthetase (3), and dimethyl sulfoxide has been 0003-2697/79/090152-03$02.00/O
Riverside,
MATERIALS
AND METHODS
Reagents. Chorismic acid (barium salt) was purchased from Sigma Chemical Company, St. Louis, Missouri. Glutamine, dithiothreitol, and bovine serum albumin (BSA)l were also from Sigma. Ethyl acetate was Fisher spectral grade, from Fisher Scientific Company, Chemical Manufacturing Division, Fair Lawn, New Jersey. Other reagents were reagent grade or better. Stock solutions of the assay reagents were prepared to the following concentrations in distilled water: glutamine, 0.2 M; MgSOd, 0.2 M; Tris-HCl buffer, pH 8.2, 0.2 M; dithiothreitol, 10 mM; and barium chorismate, 5 rnr+I (stored frozen). Cells and enzyme preparations.
E. coli,
strain Wl485 tna- (lacking tryptophanase), was a generous gift from Professor Oliver Smith. Cells were grown in Vogel and Banner minimal salts medium (5) plus 0.5% glucose at 37’C. All experiments were done ’ Abbreviation 152
used:
BSA,
bovine
serum
albumin.
ANTHRANILATE
SYNTHETASE
with log-phase cells. Crude sonic extracts were prepared in order to compare the efficacy of chemical vs physical decryptification. Log-phase cells were harvested by centrifugation and resuspended in a small volume of assay buffer containing all components except chorismate. The cells were sonicated for a total of 2 min in 20-s bursts with a Bronwill Biosonik sonicator set at 70% of full power. Extracts prepared in this way were used immediately, without further processing. Cell-free extracts of anthranilate synthetase were prepared in an essentially similar manner, except that the cells were derepressed by addition of 7-methyl indole to a final concentration of 100 pg/ml(6) for 2 h prior to harvesting, and the sonic extract was clarified by centrifugation. The supernatant was dialyzed to remove 7methyl indole and stored frozen in l.O-ml aliquots . Standard assay. A reaction buffer was prepared from the reagent stock solutions by mixing the following amounts per individual assay: glutamine, 0.2 ml; MgSOa, 0.1 ml; dithiothreitol, 0.1 ml; barium chorismate, 0.1 ml; Tris-HCl buffer, 0.1 ml; and distilled water, 0.4 ml. This buffer was dispensed (0.9 ml) into 13 x lOO-mm disposable culture tubes, and 1.5 ml of spectral-grade ethyl acetate was added per tube. The tubes were then prewarmed in a 37’C water bath for 5 min. The reaction was started by adding 100 ~1 of cell suspension, usually directly from liquid culture, and the tubes were immediately vortex for about 1 s. The tubes were then capped with metal culture tube closures and incubated in a slanted position in a shaker-water bath. Incubation was at 37’C with moderate shaking. At precisely 30 min, the reaction was stopped by addition of 0.2 ml of 1 M potassium phosphate buffer, pH 1.5. The tubes were vigorously vortexed for 10 s each and then centrifuged at low speed for 10 min to separate the two phases. A portion of the ethyl acetate layer was removed with a disposable Pasteur pipet, and the fluorescence of the extracted anthranilic acid was read in
153
ASSAY
an Aminco-Bowman spectrofluorometer (excitation, 350 nm; emission, 400 nml. RESULTS
AND DISCUSSION
E. c& anthranilate synthetase activity is not entirely immune to the presence of ethyl acetate. When cells or cell-free extracts were preincubated in the presence of ethyl acetate in reaction mixtures lacking chorismate, and the reaction subsequently started by its addition, there was a progressive loss of activity with preincubation time. Extracts lost activity at about twice the rate of whole’cells. This preincubation loss was minimized by increasing glutamine concentration up to about 0.04 M and MgSOJ concentration up to 0.02 M. (The MgSOd causes the potentially troublesome barium to precipitate as the insoluble sulfate.) Additional protection for extracts was afforded by BSA, up to 5-10 rndrnl, but this was much less effective for whole cells. Glycerol was virtually ineffective at concentrations which did not produce apparent inhibition (O-5% glycerol). Chelating agents such as EDTA and ethylene glycol bis@aminoethyl ether) N,N’-tetraacetic acid (EGTA) were also ineffectual. The composition of the standard reaction buffer was designed in part on the basis of these observations. Whole cells preincubated for 15 min in the standard buffer lost approximately 27% of the enzyme activity observed in cells which were not preincubated. When cells were preincubated in the presence of chorismate, but not glutamine, and the reaction started by addition of the latter, the activity lost in 15 min was 18%. However, the standard assay was linear with incubation time (Fig. la), which indicates that significant inactivation does not occur when both substrates are present. It is apparent that errors due to this inactivation may be introduced if the reaction is started by addition of substrate, rather than by addition of cells. The standard assay was linear with number of cells over a broad range of added cells (Fig. lb). It is convenient to use 3 to 10 x 10’
154
RICHARD
z~
/
D. BLISS
l
;
750..
.
L
/
5
5 : :
: 500.. u
s 2 ii
/
./
E
250..
: /
l/
30 L5 Time lh41nulesl
./
: i2
/
/ OO I5
2
60
./ 2m / 1, ‘:. 5
6 1 Log No Cells
s
FIG. 1. (a) The increase in anthranilate fluorescence versus time for the standard assay condition. All tubes were started simultaneously by addition of 3 x 10’ cells directly from liquid culture. Tubes were removed from the shaker-water bath at the times indicated and analyzed for anthranilate fluorescence as described under methods. (b) The increase in anthranilate fluorescence versus number of cells added to the standard assay. The reaction was started by addition of 100 ~1 of cells diluted in growth medium to the appropriate titer.
cells per assay, which yields adequate precision (23%) and accuracy, although derepressed activity can be detected in as few as 105 cells. When anthranilic acid was added to the standard assay, recovery was 98-100%. When anthranilate was added in the absence of chorismate, there was no loss of fluorescence, indicating that the enzyme product was not lost by conversion to other intermediates in the tryptophan pathway. When crude sonic extracts were assayed by the same protocol, the whole-cell method yielded 10% higher activity per cell. When ethyl acetate was added at the end of the incubation period, whole-cell assays yielded about 10% of the fluorescence exhibited by the standard procedure. Thus, the presence of ethyl acetate is essential to efficient decryptification. Crude sonic extracts yielded similar final fluorescence values, whether ethyl acetate was added at the beginning or termination of incubation. For some experimental situations, it is desirable to arrest growth and enzyme synthesis before assaying for specific activity. When lOO-~1 aliquots of cell suspensions containing 0.01 M sodium azide and 100
pg/ml chloramphenicol were assayed as described, apparent enzyme inhibition was less than 2%. When these compounds were added directly to the reaction mixture to final concentrations of 0.01 M azide and 100 pg/ml chloramphenicol, inhibition increased to about 8%. Since these latter amounts correspond to 10 times the concentration normally required to stabilize enzyme-specific activity, interference by these materials should not be significant in most cases. REFERENCES 1. Creighton, T. E., and Yanofsky, C. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17A, pp. 365-380, Academic Press, New York/London. 2. Egan, A. F., and Gibson, F. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17A, pp. 380-386, Academic Press, New York/London. 3. Blundell, M., Craig, E., and Kennell, D. (1972) Nature
New
Biol.
238, 46-4.
4. Fantes, P. A., Roberts, L. M., and Huetter, R. (1976) Arch. Microbid. 107, 207-214. 5. Vogel, H., and Banner, D. M. (1956) .I. Biol. Chem. 218, 97- 106. 6. Held, W. A., and Smith, 0. H. (197O)J. Bacteriol. 101, 209-217.
BIOCHEMISTRY
A Simple
96,
152-154
Whole-Ceil
(1979)
Assay for E. co/i Anthranilate
Synthetase
RICHARD D. BLISS Department
of Biology,
University Received
of Cai$ornia, September
Copytight 0 1979 by Academic Press. Inc. All tights of reproduction in my form reserved.
California
92521
29, 1978
used successfully to decryptify the same enzyme in yeast (4). The method reported here combines decryptification and batch assay of the E. coii enzyme in a single step. The assay is a modification of the technique of Egan and Gibson (2), who used ethyl acetate to extract the product anthranihc acid from an acid-stopped reaction mixture. I have found that the ethyl acetate may also be used to decryptify the enzyme in whole cells, and, thus, the same reagent may be used to perform two functions.
Anthranilate synthetase, the first enzyme of the tryptophan biosynthetic pathway, is usually assayed in cell-free extracts by determining the rate of appearance of the fluorescent product, anthranilic acid (1,2). The reaction is usually carried out in a spectrofluorometer cuvette while it is in the instrument, and the increase in product fluorescence is recorded on a strip chart. While this assay has proved to be useful in studies of the enzyme’s properties, it is cumbersome for derepression experiments and other studies in which intracellular enzyme specific activity is the parameter of interest. Since the assays must be carried out sequentially, substantial amounts of time may be involved in preparing reaction mixtures, rinsing cuvettes, and recording the data. Furthermore, preparation of a large number of cell-free extracts, either by sonication or other physical means, adds considerably to the labor per assay. Specific activity measurements may be simplified by the use of stopped assays, which permit the reaction to be carried out for several samples simultaneously. A further simplification is possible through the use of a whole-cell assay, in which the cells are treated chemically to decryptify the enzyme activity, thus avoiding the necessity for physical disruption. Suitable methods of decryptification vary with the enzyme activity under investigation, but treatment with a mixture of lysozyme, detergent, and ethylenediaminetetraacetate (EDTA) has been reported as being satisfactory for use with Escherichia coli anthranilate synthetase (3), and dimethyl sulfoxide has been 0003-2697/79/090152-03$02.00/O
Riverside,
MATERIALS
AND METHODS
Reagents. Chorismic acid (barium salt) was purchased from Sigma Chemical Company, St. Louis, Missouri. Glutamine, dithiothreitol, and bovine serum albumin (BSA)l were also from Sigma. Ethyl acetate was Fisher spectral grade, from Fisher Scientific Company, Chemical Manufacturing Division, Fair Lawn, New Jersey. Other reagents were reagent grade or better. Stock solutions of the assay reagents were prepared to the following concentrations in distilled water: glutamine, 0.2 M; MgSOd, 0.2 M; Tris-HCl buffer, pH 8.2, 0.2 M; dithiothreitol, 10 mM; and barium chorismate, 5 rnr+I (stored frozen). Cells and enzyme preparations.
E. coli,
strain Wl485 tna- (lacking tryptophanase), was a generous gift from Professor Oliver Smith. Cells were grown in Vogel and Banner minimal salts medium (5) plus 0.5% glucose at 37’C. All experiments were done ’ Abbreviation 152
used:
BSA,
bovine
serum
albumin.
ANTHRANILATE
SYNTHETASE
with log-phase cells. Crude sonic extracts were prepared in order to compare the efficacy of chemical vs physical decryptification. Log-phase cells were harvested by centrifugation and resuspended in a small volume of assay buffer containing all components except chorismate. The cells were sonicated for a total of 2 min in 20-s bursts with a Bronwill Biosonik sonicator set at 70% of full power. Extracts prepared in this way were used immediately, without further processing. Cell-free extracts of anthranilate synthetase were prepared in an essentially similar manner, except that the cells were derepressed by addition of 7-methyl indole to a final concentration of 100 pg/ml(6) for 2 h prior to harvesting, and the sonic extract was clarified by centrifugation. The supernatant was dialyzed to remove 7methyl indole and stored frozen in l.O-ml aliquots . Standard assay. A reaction buffer was prepared from the reagent stock solutions by mixing the following amounts per individual assay: glutamine, 0.2 ml; MgSOa, 0.1 ml; dithiothreitol, 0.1 ml; barium chorismate, 0.1 ml; Tris-HCl buffer, 0.1 ml; and distilled water, 0.4 ml. This buffer was dispensed (0.9 ml) into 13 x lOO-mm disposable culture tubes, and 1.5 ml of spectral-grade ethyl acetate was added per tube. The tubes were then prewarmed in a 37’C water bath for 5 min. The reaction was started by adding 100 ~1 of cell suspension, usually directly from liquid culture, and the tubes were immediately vortex for about 1 s. The tubes were then capped with metal culture tube closures and incubated in a slanted position in a shaker-water bath. Incubation was at 37’C with moderate shaking. At precisely 30 min, the reaction was stopped by addition of 0.2 ml of 1 M potassium phosphate buffer, pH 1.5. The tubes were vigorously vortexed for 10 s each and then centrifuged at low speed for 10 min to separate the two phases. A portion of the ethyl acetate layer was removed with a disposable Pasteur pipet, and the fluorescence of the extracted anthranilic acid was read in
153
ASSAY
an Aminco-Bowman spectrofluorometer (excitation, 350 nm; emission, 400 nml. RESULTS
AND DISCUSSION
E. c& anthranilate synthetase activity is not entirely immune to the presence of ethyl acetate. When cells or cell-free extracts were preincubated in the presence of ethyl acetate in reaction mixtures lacking chorismate, and the reaction subsequently started by its addition, there was a progressive loss of activity with preincubation time. Extracts lost activity at about twice the rate of whole’cells. This preincubation loss was minimized by increasing glutamine concentration up to about 0.04 M and MgSOJ concentration up to 0.02 M. (The MgSOd causes the potentially troublesome barium to precipitate as the insoluble sulfate.) Additional protection for extracts was afforded by BSA, up to 5-10 rndrnl, but this was much less effective for whole cells. Glycerol was virtually ineffective at concentrations which did not produce apparent inhibition (O-5% glycerol). Chelating agents such as EDTA and ethylene glycol bis@aminoethyl ether) N,N’-tetraacetic acid (EGTA) were also ineffectual. The composition of the standard reaction buffer was designed in part on the basis of these observations. Whole cells preincubated for 15 min in the standard buffer lost approximately 27% of the enzyme activity observed in cells which were not preincubated. When cells were preincubated in the presence of chorismate, but not glutamine, and the reaction started by addition of the latter, the activity lost in 15 min was 18%. However, the standard assay was linear with incubation time (Fig. la), which indicates that significant inactivation does not occur when both substrates are present. It is apparent that errors due to this inactivation may be introduced if the reaction is started by addition of substrate, rather than by addition of cells. The standard assay was linear with number of cells over a broad range of added cells (Fig. lb). It is convenient to use 3 to 10 x 10’
154
RICHARD
z~
/
D. BLISS
l
;
750..
.
L
/
5
5 : :
: 500.. u
s 2 ii
/
./
E
250..
: /
l/
30 L5 Time lh41nulesl
./
: i2
/
/ OO I5
2
60
./ 2m / 1, ‘:. 5
6 1 Log No Cells
s
FIG. 1. (a) The increase in anthranilate fluorescence versus time for the standard assay condition. All tubes were started simultaneously by addition of 3 x 10’ cells directly from liquid culture. Tubes were removed from the shaker-water bath at the times indicated and analyzed for anthranilate fluorescence as described under methods. (b) The increase in anthranilate fluorescence versus number of cells added to the standard assay. The reaction was started by addition of 100 ~1 of cells diluted in growth medium to the appropriate titer.
cells per assay, which yields adequate precision (23%) and accuracy, although derepressed activity can be detected in as few as 105 cells. When anthranilic acid was added to the standard assay, recovery was 98-100%. When anthranilate was added in the absence of chorismate, there was no loss of fluorescence, indicating that the enzyme product was not lost by conversion to other intermediates in the tryptophan pathway. When crude sonic extracts were assayed by the same protocol, the whole-cell method yielded 10% higher activity per cell. When ethyl acetate was added at the end of the incubation period, whole-cell assays yielded about 10% of the fluorescence exhibited by the standard procedure. Thus, the presence of ethyl acetate is essential to efficient decryptification. Crude sonic extracts yielded similar final fluorescence values, whether ethyl acetate was added at the beginning or termination of incubation. For some experimental situations, it is desirable to arrest growth and enzyme synthesis before assaying for specific activity. When lOO-~1 aliquots of cell suspensions containing 0.01 M sodium azide and 100
pg/ml chloramphenicol were assayed as described, apparent enzyme inhibition was less than 2%. When these compounds were added directly to the reaction mixture to final concentrations of 0.01 M azide and 100 pg/ml chloramphenicol, inhibition increased to about 8%. Since these latter amounts correspond to 10 times the concentration normally required to stabilize enzyme-specific activity, interference by these materials should not be significant in most cases. REFERENCES 1. Creighton, T. E., and Yanofsky, C. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17A, pp. 365-380, Academic Press, New York/London. 2. Egan, A. F., and Gibson, F. (1970) in Methods in Enzymology (Tabor, H., and Tabor, C. W., eds.), Vol. 17A, pp. 380-386, Academic Press, New York/London. 3. Blundell, M., Craig, E., and Kennell, D. (1972) Nature
New
Biol.
238, 46-4.
4. Fantes, P. A., Roberts, L. M., and Huetter, R. (1976) Arch. Microbid. 107, 207-214. 5. Vogel, H., and Banner, D. M. (1956) .I. Biol. Chem. 218, 97- 106. 6. Held, W. A., and Smith, 0. H. (197O)J. Bacteriol. 101, 209-217.
