Fluorescent assay of anthranilate synthetase-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzyme-complex on polyacrylamide gels

ANALYTICAL

BIOCHEMISTRY

6.5,458-465

( 1975)

Fluorescent Assay of Anthranilate 5Phosphoribosylpyrophosphate Enzyme-Complex THOMAS Biological

Research

H. GROVE Laboratories, Syracuse.

Synthetase-Anthranilate Phosphoribosyltransferase

on Polyacrylamide AND

H. RICHARD

Department New York

of Biology8 13210

Gels LEVY Syracuse

University,

Received October 14. 1974: accepted December 11, 1974 A rapid and sensitive method for the localization of anthranilate synthetaseanthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase enzymecomplex from Salmonella typhimvrium after disc gel electrophoresis has been developed, based on the fluorescence of the anthranilate synthetase product. anthranilate. Both NH:,-and glutamine-dependent anthranilate synthetase activities could be detected. A linear transport attachment on a Gilford Spectrophotometer was modified to scan the fluorescent gels. Semiquantitative evaluation of enzyme-complex activity was achieved by interpreting the chart recorder tracings of assayed gels. The enzyme-complex on polyacrylamide gels can be assayed under various conditions. The concentration of tryptophan required for half-maximal inhibition of NH,-dependent anthranilate synthetase activity was determined from enzyme-complex assayed on polyacrylamide gels and in solution. A value of 4-8 PM tryptophan was determined in both cases. The technique is applicable to any enzyme that forms a fluorescent product.

In the course of our efforts to purify and characterize normal and mutant anthranilate synthetase (AS)-anthranilate 5-phosphoribosylpyrophosphate phosphoribosyltransferase (PRT) enzyme-complex from Salmonella typhimurium, we found it necessary to develop a procedure for the visualization of activity on polyacrylamide gels after disc gel electrophoresis. Use was made of the natural fluorescence of anthranilate, the product of the AS reaction. When AS is not associated with PRT, only NH, can be used as the amine donor in the AS reaction. Association of AS with PRT allows either NH3 or glutamine to be utilized as a substrate (1): chorismate + NH3 (or L-glutamine) --+ anthranilate + pyruvate + H’ + (L-glutamate). By allowing the reaction to proceed in the presence of either glutamine or NH, and then observing the fluorescence of the anthranilate produced, the location of associated or unassociated AS can be detected on polyacrylamide gels. The procedure was made continuous and semi458 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

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quantitative by adapting a Gilford spectrophotometer to scan the gels for anthranilate fluorescence (2). The reaction velocities can be determined for the enzyme-complex under various conditions and thus the enzyme-complex can also be characterized on polyacrylamide gels. MATERIALS

AND

METHODS

Chemicals. Barium chorismate, L-glutamine and triethanolamine-HCl were obtained from Sigma Chemical Co. Barium chorismate was converted to its potassium salt before use. Anthranilate, acrylamide and bisacrylamide came from Eastman Chemical Co.; ammonium persulfate from E-C Apparatus Corp.; N,N,N’,N’-tetramethylethylenediamine from Matheson, Coleman, and Bell: enzyme grade ammonium sulfate from Mann Research Labs; and Sephadex G-50 superfine from Pharmacia Fine Chemicals. All other chemicals were of reagent grade. Enzyme-complex preparation and assay. Enzyme-complex from S. typhimurium trp E2 was purified to 93% homogeneity by classical techniques and stored as described elsewhere (3,4). The enzyme-complex was gel-filtered immediately before use on a precalibrated column (0.5 X 6.5 cm) of Sephadex G-50 superfine. Only a small volume of protein solution, 20 ~1, was gel-filtered at a time. The column was developed with 0.1 M potassium phosphate buffer, pH 7.4 containing 0.1 mM ethylenediaminetetraacetate and 0.4 mM 2-mercaptoethanol. Ammoniadependent AS activity in solution was assayed as previously described (3). These assays were performed with a Hitachi Perkin-Elmer MPF-3 Spectrofluorimeter equipped with a thermostated sample compartment maintained at 25” (excitation 320 nm; emission 400 nm). Ammonia and glutamine-dependent AS activities on polyacrylamide gels were assayed using reaction mixtures modified from that of Zalkin and Kling (5) and Henderson et al. (4), respectively. The final concentrations of the reactants for measuring NH,-dependent AS activity were: 0.34 mM chorismate, 50 mM ammonium sulfate, 5 mM MgCI,, 2 mM 2-mercaptoethanol and 50 mM triethonolamine-HCl buffer, pH 8.3; for glutamine-dependent AS activity they were: 0.34 mM chorismate, 5 mM glutamine, 2.5 mM MgCl, and 50 mM potassium phosphate buffer. pH 7.4. Protein was determined by a microbiuret procedure (6). Electrophoresis. Disc gel electrophoresis was performed according to Davis (7) on 5% polyacrylamide with a Tris-glycine pH 8.3 reservoir buffer. Preparation of gels and electrophoresis were done at room temperature. The gels, cast in 0.5 X 7.5 cm glass tubes, were pre-run at 2.5 mA per gel for 30 min. No spacer gel was used. Instead, a dense sample was formed by adding one drop of glycerin to every eight drops of gelfiltered protein solution. The sample was then layered directly on the separating gel. Bromophenol blue tracking dye was included in the upper

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reservoir buffer in order to determine the time necessary for the initial run of 1.25 mA per gel (found to be 20 min) and the secondary run of 2.5 mA per gel (45 min). Protein on the gels was stained using Coomassie brilliant blue in order to determine a R, for the enzyme-complex. After the conditions were determined Bromophenol blue was no longer used when the gels were to be assayed for AS activity. The electrophoresed gels were retained in their tubes until scanned. Scanning procedure. Gels were removed from the electrophoresis tubes and immediately placed in a quartz boat. The boat was aligned in a Gilford 240 Spectrophotometer equipped with a linear transport attachment and 2 ml of assay solution were added. The gels were scanned at a speed of 4 cm/min after various times of incubation in the assay solutions. The velocity of the enzyme-complex catalyzed reaction is expressed as the increase in the inverted peak height in centimeters per minute. Reaction velocities can also be measured by observing the continuous increase in fluorescence with the active enzyme-complex positioned in front of the photometer aperture. The Gilford Spectrophotometer was modified (2) to measure anthranilate fluorescence by replacing the linear transport lid with a Long Wave UVL-21 Blak-Ray Lamp (Ultra-Violet Products. Inc., San Gabriel, CA) and inserting a Wratten 2B filter between the quartz boat and photometer. A black cloth was used to cover the entire sample compartment assembly. There was no temperature control system and some heat was generated during the assay. The wavelength was adjusted to 325 nm and the slit width to 0.25 mm. The “read/blank” switch was set on “blank” and the pen set to full scale deflection using the zero adjust and then the vernier knob, if necessary. The digital absorbance setting did not affect the fluorescent reading. However, the fluorescent scale can be proportionally altered by changing the ratio mode setting. Since the fluorescent signal is linear with percent transmission and not with the absorbance measured by the Gilford Spectrophotometer, the signal can be quantitated only by determining the antilogarithm of the inverted peak height on the chart recorder tracing. Quantitation of fluorescence was simplified by assuming that linearity existed between the fluorescent signal and anthranilate concentration for the first 40% of the scale. This was verified by measuring a range of anthranilate concentrations in the modified Gilford spectrophotometer. All the measurements described in this paper were done within this range. RESULTS AND

DISCUSSION

Time sequences of fluorescent tracing of NH,- and glutamine-dependent AS activities on polyacrylamide gels are shown in Fig. 1. Because the Gilford Spectrophotometer measures absorbance, the fluorescent

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t 1. Representative tracings of fluorescent gels scanned by the modified Gilford Spectrophotometer. The gel assays and scanning procedure are described in Materials and Methods. The time scale at the right refers to time of incubation. There were 1 I pg of protein on each gel. (A) NH,-dependent AS activity. The ratio mode was set at 1.0. (B) Glutamine-dependent AS activity. The ratio mode was set at 0.5. FIG.

signal appears as a trough, or negative absorbance. The fluorescent bands are broad but a peak is clearly distinguishable. It would be difficult to measure activity of two or more closely spaced enzymecomplex bands because of this peak width. The enzyme-complex protein band overlayed the enzyme-complex activity band. The RM for enzymecomplex assayed on polyacrylamide gel for NH,- or glutamine-dependent AS activity was 0.24 in both cases. The RM for enzyme-complex on polyacrylamide gel stained for protein was 0.27. The rates measured were linear over the time scale used (Fig. 2). This permitted determination of the velocity of the enzyme-complex catalyzed reactions on the polyacrylamide gels. Gels may be assayed up to 1.5 h after electrophoresis without any appreciable decrease in activity. Storing the gels before assay between 0 and 4” will slow down the diffusion of the enzyme-complex. Inspection of assayed gels (Fig. 3) revealed one major fluorescent band with enzyme-complex activity and a very weak fluorescent band at the buffer front with no enzyme-complex activity. Both bands appeared pale violet. The buffer front band was observed in gels electrophoresed without protein; it was not detected by the modified Gilford Spectropho-

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0 IO TIME OF INCUBATION

20 (MINUTES)

FIG. 2. Rate of NH,- and glutamine-dependent AS activities on polyacrylamide gels. The data were derived from Fig. 2. The first tracing in both sets served as the baseline for measuring the inverted peak heights. The inverted peak height values for the glutaminedependent AS reaction were adjusted to the same scale as the NH,-dependent AS reaction peaks: (0) glutamine-dependent AS activity: (0) NH,-dependent AS activity.

FIG. 3. Fluorescent gels. The gels were assayed as described in Materials and Methods for IO min and then place on a black cloth. Side lighting was provided by two long wave uv lights. Corning No. 18 filters and a Wratten 2B filter were used for photography. High speed daylight Ektachrome color slide film was used at f5.6 for 8 s. Black and white prints were made from the developed slide by Mr. Richard Russell, Rochester, NY. (A) NH,dependent AS assayed gel; (B) glutamine-dependent AS assayed gel.

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tometer. It was probably fainter than that observed by Ragland er al. (2) due to the use of a different filter and pre-electrophoresis in our system. The origin of this buffer front band is not clear but apparently the band is due to fluorescent contaminants in the gel reagents. The reaction mixture is specific for the enzyme-complex. as demonstrated by the absence of fluorescence produced when the substrate chorismate was not included in the NH,- or glutamine-dependent AS assay solution or when gels with 11 pg of electrophoresed protein were heated at 75” for 10 min and then assayed for NH,- or glutamine-dependent AS activity. When these gels, or gel minus protein, were scanned for fluorescence, the pattern resembled that of the first tracings in Fig. 1A or B. The assayed gels may be stored by quick-freezing on dry ice and keeping the frozen gels in a sealed glass tube at -64” in the dark. An NH,-dependent AS assayed gel has been kept 40 days without peak broadening or displacement. The rate of NH,-dependent AS activity was determined for several protein concentrations (Fig. 4). The rates were propo~ion~ to the protein concentration from 1 to 53 pg. There was a large amount of scatter in peak heights when using less than 10 pg of protein per gel (0.029 IU of NH,-dependent AS activity) and rates were difficult to measure. With low amounts of enzyme-complex, diffusion becomes significant and antagonistic to the fluorescent assay procedure. The usefulness and sensitivity of the fluorescent assay procedure was tested by determining the concentration of tryptophan required for half-

,ug PROTEIN

PER GEL

FIG. 4. Dependence of the fluorescent assay procedure on protein concentration. The NH,-dependent AS gel assay is described in Materials and Methods except the concentration of chorismate was 68 pM.

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0 100 L-TRYPTOPHAN

200

300

(PM)

FIG. 5. Inhibition by tryptophan of NH,-dependent AS solution and on polyacrylamide gels. The assays in solution described in Materials and Methods. There were 21 pg of gel: (0) enzyme-complex in solution; (0) enzyme-complex

activity of enzyme-complex in and on polyacrylamide gels are protein on each polyacrylamide on polyacrylamide gels.

maximal inhibition of NH,-dependent AS activity for enzyme-complex on polyacrylamide gels and in solution (Fig. 5). In both cases, a concentration of tryptophan between 4 and 8 PM caused 50% inhibition of NH,-dependent AS activity. The fluorescent assay procedure on gels has been applied to crude preparations of enzyme-complex. Soluble enzyme-complex was pre-

FIG. 6. Tracings of fluorescent gels scanned for glutamine- and NH,-dependent AS activity. The enzyme-complex was derived from a crude extract. The gel assays and scanning procedure are described in Materials and Methods. The gels were scanned after 7 min of incubation using a ratio setting of 0.5: (A) glutamine-dependent AS activity: (B) NH,dependent AS activity.

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pared by centrifuging a sonicated suspension of S. typhimurium trp E2 cells (3,4) at 105,000 g for 3 h. Both NH3- and glutamine-dependent AS activities in 50 ~1 of supernatant (0.074 IU of NH,-dependent AS activity) were easily detected on polyacrylamide gels (Fig. 6). Thus it will be possible to compare enzyme-complexes from various mutants of Escherichia coli and S. typhimurium in electrophoretic properties and kinetics, such as tryptophan regulation, without the necessity of a complete purification. Prior resolution of enzyme-complexes by electrophoresis enables one to look at the properties of a very pure protein on a microscale. The fluorescent assay procedure may be adapted to any enzyme catalyzing a reaction in which a fluorescent product is formed. Glucose 6-phosphate dehydrogenase from human erythrocytes has been visually detected on cellulose acetate strips using the natural fluorescence of NADPH (8). Such enzymes may be assayed or located on polyacrylamide gels by the use of a fluorescent assay procedure similar to the one described in this study. Preliminary experiments with glucose 6-phosphate dehydrogenase from Leucorzostoc mesenteroides have been successful in locating enzyme activity on polyacrylamide gels by observing NADH fluorescence. ACKNOWLEDGMENT This work was supported by grant GM 19839 from the United States Public Health Service.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

Nagano, H., Zalkin, H., and Henderson, E. J. (1970). /. Biol. Chem. 245, 3810. Ragland, W. L., Pace, J., and Kemper, D. (1974) Anal. Biochem. 59, 24. Grove, T. H., and Levy, H. R. Submitted for publication. Henderson, E. J., Nagano, H., Zalkin, H., and Hwang, L. H. (1970). J. Biol. Chem. 245, 1416. Zalkin, H., and Kling, D. (1968) Biochemistry 7, 3566. Zamenhof, S. (1957) Methods Enzymol. 3, 702. Davis, B. J. (1964) Ann. N. Y. Acad. Sci. 121, 404. Sparkes, R. S., and Baluda, M. (1969) Anal. Biochem. 30, 289.