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A continual spectrophotometric assay for amino acid decarboxylases
ANALYTICAL
BIOCHEMISTRY
A Continual
170,367-371
(1988)
Spectrophotometric
Assay for Amino Acid Decarboxylases
FIONA SCRIVEN, KENNETH B. WLASICHUK, AND MONICA M. PALCIC’ Department of Food Science, University of Alberta, Edmonton, Alberta. Canada T6G 2P5 Received October 1, 1987 A spectrophotometric method for assaying the activity of three amino acid decarboxylases is reported. This method makes use of the coupled reaction of the decarboxylase with phosphoenolpyruvate carboxylase and malate dehydrogenase. The assay is simple and rapid and allows continuous monitoring of the reaction progress. The kinetic parameters obtained using this method for diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase are comparable to values obtained by radiochemical methods. 8 1988 Academic mess, hc. KEY WORDS: diaminopimelate decarboxylase; lysine decarboxylase; atginine decarboxylase; continuous spectrophotometric assay.
The activity of amino acid decarboxylases is most commonly estimated by measuring CO2 evolution manometrically or by trap ping and counting 14C02 from carboxyl-labeled amino acid substrates (l-4). The manometric method yields continual data; however, this procedure is generally tedious, insensitive, and cumbersome. The radiochemical procedures are sensitive, but require a panel of appropriately labeled substrates, and they are time consuming and do not provide continual monitoring of the reaction course. Other methods based on the isolation and quantitation of the amine products (5-7) or their coupling to a second enzyme, for example, an amine oxidase (3,8), have been reported for individual decarboxylases but are not of general use, with the exception of a CO2 selective electrode reported for lysine decarboxylase (9). In this paper we report on a general spectrophotometric method which couples the formation of CO2 to decreases in NADH absorbance by phosphoenolpyruvate carboxylase and malate dehydrogenase. The sequence of the reactions is ’ To whom all correspondence should be addressed.
367
Amino Acid Decarboxylase Amine + CO2
[I]
CO* + Hz0 + Phosphoenolpyruvate
Phosphoenolpyruvate
-
carlxxylase
Oxaloacetate + H2P0; Oxaloacetate + H+
Malate
[2]
+ NADH Dehydrogenax
+ NAD + Malate.
[3]
The velocity of the decarboxylation reaction is estimated from the rate of oxidation of NADH. The method is rapid, reproducible, and accurate and has been used to estimate diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase activity, but in principle it can be applied to any decarboxylase which is active in the pH range 6.6 to 7.7. MATERIALS
AND METHODS
L-Arginine hydrochloride, L-arginine decarboxylase (Type IV, from Escherichia coli, 320 units/mg), D,L-diaminopimelic acid, Llysine monohydrochloride, L-lysine decarboxylase (Type VIII, from Bacterium cadaveris, 55 units/mg), pyridoxal 5’-phosphate, and the carbon dioxide diagnostic kit were
0003-2697188 $3.00 Copyright 0 1988 by Academic Ress. Inc. All rights of production in any form reserved.
368
SCRIVEN, WLASICHUK,
from the Sigma Chemical Co. ACS scintillation cocktail, L-[U-‘4C]lysine (300 mCi/ mmol), L-[U-‘*C]arginine (342 mCi/mmol), and [ 1,7-14C]diaminopimelic acid (50 mCi/ mmol) were from Amersham. All other chemicals were of reagent grade. Diaminopimelate decarboxylase was isolated from Bacillus sphaericus IF0 3525 by the method of Asada et al. ( IO) and had a specific activity of 0.56 units/mg protein when assayed using the radiochemical method described below with 1.2 mM diaminopimelate in 50 mM potassium phosphate buffer at pH 6.8 (11). Protein was estimated using the Bio-Rad protein assay, which is based on the method described by Bradford ( 12), using bovine serum albumin as a protein standard. A Beckman DU-8 spectrophotometer thermostatted to 37°C was used for optical measurements. SPECTROPHOTOMETRIC
ASSAY
The carbon dioxide diagnostic kit has two components: a CO, free aqueous diluent and CO2 reagent vials that, upon reconstitution with 6.5 ml of diluent, contain 1.79 mM phosphoenolpyruvate, 0.4 mM NADH, phosphoenolpyruvate carboxylase (0.2 12 units/ml), and malate dehydrogenase ( 1.25 units/ml) in Tris buffer, pH 7.7, at 37°C. This assay solution was used for lysine decarboxylase and diaminopimelate decarboxylase assays. A background rate was estimated by adding substrate (in up to 0.1 ml) to 1 ml of reconstituted solution and monitoring the change in absorbance at 340 nm. Typical background rates are 0.0005 AOD/min. The reaction was then initiated by the addition of enzyme in volumes of up to 50 ~1. The addition of enzyme to the kit components gave no change in absorbance above background, unless substrate was present. For assays at lower pH values, the kit was reconstituted with the COZ free aqueous diluent and the pH adjusted with 0.75 M phosphoric acid ( 1OO- 115 ~1) to give a Tris-phos-
AND PALCIC
phate buffer in the pH range 6.6 to 6.8. In initial trials, the lyophilized kit components were reconstituted with 6.5 ml of 50 mM KH2P04 acidified with phosphoric or sulfuric acid. In this case the buffer component is degassed under vacuum for 10 min prior to use in reconstituting the kit. RADIOCHEMICAL
ASSAY
Diaminopimelate decarboxylase was assayed by monitoring 14C02 evolution from [ 1,7-‘4C]diaminopimelate. Assay mixtures contained 50 mM potassium phosphate buffer or 100 mM Tris buffer at the appropriate pH, substrate, 0.25 PC1 of labeled diaminopimelate, and enzyme in a total volume of 0.86 ml in a scintillation vial. The caps of the vials contained a 1.5 X 1.5~cm piece of filter paper impregnated with 20 ~1 of 1 M hyamine hydroxide solution as a 14C02 trap ping agent. The reaction was initiated by the addition of enzyme and the assay mixture was incubated at 37°C for 20 min with continual shaking. The reaction was terminated by the addition of 0.2 ml of 10% trichloroacetic acid and the vials were shaken for 60 min to ensure that 14C02 evolution was complete. The filter papers were removed and counted in 10 ml of ACS scintillation cocktail on a Beckman LS 180 1 scintillation counter. Lysine decarboxylase was assayed in 100 mM Tris-phosphate buffer, pH 6.7, containing L-lysine, 0.14 rCi labeled L-lysine, and enzyme in a total volume of 1.O ml in a scintillation vial. Following incubation at 37°C for 15 min the reaction was quenched with 0.2 ml of 10% trichloroacetic acid and 14C02 was trapped as described for diaminopimelate decarboxylase. Arginine decarboxylase was assayed in an analogous fashion in 100 mM Tris-phosphate buffer, pH 6.6, containing substrate, 0.13 &i labeled L-arginine, and enzyme in 1.O ml. Incubations were carried out for 10 min at 37”C, quenched, and
A SPECTROPHOTOMETRIC
treated as described for diaminopimelate carboxylase.
369
ASSAY FOR DECARBOXYLASES
de-
RESULTS AND DISCUSSION
The coupled assay was proportional to diaminopimelate decarboxylase concentration, as seen in Fig. 1, where a linear relationship with a correlation coefficient of 0.998 was obtained between AOD/min at 340 nm and enzyme concentration. These absorbance changes correspond to the range of 4.9 to 39 nmol product/min, using a millimolar extinction coefficient of 6.22 mM-’ cm-’ for NADH absorbance at 340 nm. The lower limits that can be quantitated are 1.5 nmol product/min, which corresponds to a change of 0.008 OD/min. The reaction is linear for at least 10 min for concentrations of enzyme giving 0.15 AOD/min or less. At higher concentrations, rapid depletion of NADH causes curvature in 3 to 7 min, but initial rates can still be estimated from tangents to the curves at maximal slopes. The effect of substrate concentration on the initial rate of the diaminopimelate decarboxylase-catalyzed reaction at pH 7.7 is
0.2 -
.G E
75
pg Diaminopimelate
Decarboxylase
FIG. 1. The effect of enzyme concentration on the reaction rate. Diaminopimelate decarboxylase isolated from Bacillus sphaericus was used. The final diaminopimelate concentration was 10 mM, and measurements were carried out in a total volume of 1.15 ml at pH 7.7 and 37’C.
1
OO
5
l/[Diaminopimelate]
I 15
10
(mM-‘)
FIG. 2. The effect of diaminopimelate concentration on the rate of CO* production. The activity was estimated spectrophotometrically at 340 nm in a volume of 1.15 ml at pH 7.7 and 37’C using 25 pg of enzyme.
shown as a reciprocal plot in Fig. 2. These data were analyzed using a computer program based on the statistical method of Wilkinson (13) and gave a V,, of 0.54 f 0.02 pmol/min/mg of protein with a K,,, of 0.67 f 0.06 mM. When the radiochemical method was employed at this pH, values of 0.66 -t 0.06 pmol/min/mg and 1.O + 0.2 mM were obtained for V,,, and Km, respectively. Though the reasons for the discrepancies in these values is unclear, we do not consider these to be major, in view of the great differences in the two methods used. The diaminopimelate decarboxylase assay was also carried out at pH 6.8. For this pH a K,,,of 2.1 f 0.2 mM was obtained for the spectrophotometric assay and 2.3 10.3 mM for the radiochemical method; these values are comparable to 1.8 mM reported by Asada et al. ( 10). The values of V,, are 0.78 f 0.04 and 0.84 * 0.04 pmol/min/mg for the spectrophotometric and radiochemical methods, respectively. Lysine decarboxylase was assayed spectrophotometrically at pH 6.7 and again the dependence of the initial rate upon enzyme concentration was linear with a correlation coefficient of 0.998 (Fig. 3). The effect of lysine concentration on reaction velocity at pH 6.7 is shown as a reciprocal plot in Fig. 4. Analysis of these data using the Wilkinson
370
SCRIVEN, WLASICHUK,
method gave a V,, of 48 + 1pmol/min/mg. Substrate inhibition was observed for lysine concentrations greater than 5 mM (data not plotted). The K, obtained by fitting the initial rate data below this concentration is 0.36 + 0.03 mM. The radiochemical assayat pH 6.7 gave V,, 45 + 7 rmol/min/mg and K,,, 0.42 + 0.04 mM. When arginine decarboxylasewas assayed spectrophotometrically at pH 6.6, a V,,,,,of 75 f 6 pmol/min/mg and a K, of 1.2 + 0.2 mM were obtained. The corresponding values obtained with the radiochemical assay are 71 + 5 rmol/min/mg and 1.Of 0.2 mM for I’,,,, and K,,,, respectively. The pH dependence of the arginine decarboxylasereaction has been studied (14,15) and the relative velocity is optimal from pH 5 to 6, declining sharply beyond this range. However, at pH 6.6 about 25% of the activity remains. This spectrophotometric assay could easily be employed for biosynthetic (16) or plant (3) arginine decarboxylases, which exhibit pH optima of 8 and 7.5, respectively. The overall reproducibility of the assayis good; in initial trials with lysine decarboxylasefor 20 different samplesdone in triplicate the average standard deviation was 4.7%.
AND PALCIC
l/[Lysine]
(mM-‘)
FIG. 4. The effect of lysine concentration on the rate of COz production by lysine decarboxylase at pH 6.7 and 37°C.
This included data at both high and low substrate concentrations. In summary, we report on a rapid, reproducible method for assaying three amino acid decarboxylases:diaminopimelate decarboxylase, lysine decarboxylase,and arginine deearboxylase.We find that reconstitution of a commercial kit for CO2 estimation can be conveniently employed for this purpose, although in principle the kit can be simulated by the individual reagents, which are all commercially available. The activity of these amino acid decarboxylases is reasonably comparable to that obtained using radiochemical methods. ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research.
REFERENCES
o.cm~,,,_] 0
100
ng Lyslne
zal
Decerboxylase
FIG. 3. The effect of lysine decarboxylase concentration on the reaction rate. The final lysine concentration was 5 mM and measurements were carried out in a volume of 1.15 ml at pH 6.7 and 37°C.
1. Najjar, V. A. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 3, pp. 462-464, Academic Press, New York. 2. Pegg, A. E., and McGill, S. (1979) Biochim. Biophys. Acta 568,416-427. 3. Smith, T. A. (1979) Anal. Biochem. 92, 331-337. 4. Morris, D. R., and Boeker, E. A. (1981) in Methods in Enzymology (Tabor, H. and Tabor, C. W., Eds.), Vol. 94, pp. 125-134, Academic Press, New York.
A SPECTROPHOTOMETRIC
ASSAY FOR DECARBOXYLASES
5. McCaman, M. W., McCaman, R. E., and Lees, G. J. (1972) Anal. Biochem. 45,242-252. 6. Raham, M. D. K., Nagatsu, T., and Kato, T. (1980) J. Chromalogr. 221,265-270. 7. Phan, A. P. H., Ngo, T. T., and Lenhoff, H. M. (1982) Anal. B&hem. 120, 193-197. 8. Rosei, M. A., Avigliano, L., Sabatini, S., and Rigo, A. (1984) Anal. Biochem. 139,73-76. 9. Tonelli, D., Budini, R., Gattavecchia, E., and Girotti, S. (1981) Anal. B&hem. 111, 189-194. 10. Asada, Y., Tanizawa, K., Kawabata, Y., Misono, H., and Soda, K. (198 1) Agric. Biol. Chem. 45, 1513-1514.
371
11. Kelland, J. G., Palcic, M. M., Pickard, M. A., and Vederas, J. C. (1985) Biochemistry 24, 3263-3267.
12. Bradford,
M. M. (1976) Anal. Biochem.
72,
248-254.
13. Wilkinson, G. N. (1960) B&hem. J. 80,324-332. 14. Taylor, E. S., and Gale, E. F. ( 1945) B&hem. J. 39, 52-58.
15. Blethen, S. L., Boeker, E. A., and Snell, E. E. (1968) J. Biol. Chem. 243, 1671-1677. 16. Morris, D. R., and Pardee, A. B. (1966) J. Biol. Chem. 241,3129-3135.
BIOCHEMISTRY
A Continual
170,367-371
(1988)
Spectrophotometric
Assay for Amino Acid Decarboxylases
FIONA SCRIVEN, KENNETH B. WLASICHUK, AND MONICA M. PALCIC’ Department of Food Science, University of Alberta, Edmonton, Alberta. Canada T6G 2P5 Received October 1, 1987 A spectrophotometric method for assaying the activity of three amino acid decarboxylases is reported. This method makes use of the coupled reaction of the decarboxylase with phosphoenolpyruvate carboxylase and malate dehydrogenase. The assay is simple and rapid and allows continuous monitoring of the reaction progress. The kinetic parameters obtained using this method for diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase are comparable to values obtained by radiochemical methods. 8 1988 Academic mess, hc. KEY WORDS: diaminopimelate decarboxylase; lysine decarboxylase; atginine decarboxylase; continuous spectrophotometric assay.
The activity of amino acid decarboxylases is most commonly estimated by measuring CO2 evolution manometrically or by trap ping and counting 14C02 from carboxyl-labeled amino acid substrates (l-4). The manometric method yields continual data; however, this procedure is generally tedious, insensitive, and cumbersome. The radiochemical procedures are sensitive, but require a panel of appropriately labeled substrates, and they are time consuming and do not provide continual monitoring of the reaction course. Other methods based on the isolation and quantitation of the amine products (5-7) or their coupling to a second enzyme, for example, an amine oxidase (3,8), have been reported for individual decarboxylases but are not of general use, with the exception of a CO2 selective electrode reported for lysine decarboxylase (9). In this paper we report on a general spectrophotometric method which couples the formation of CO2 to decreases in NADH absorbance by phosphoenolpyruvate carboxylase and malate dehydrogenase. The sequence of the reactions is ’ To whom all correspondence should be addressed.
367
Amino Acid Decarboxylase Amine + CO2
[I]
CO* + Hz0 + Phosphoenolpyruvate
Phosphoenolpyruvate
-
carlxxylase
Oxaloacetate + H2P0; Oxaloacetate + H+
Malate
[2]
+ NADH Dehydrogenax
+ NAD + Malate.
[3]
The velocity of the decarboxylation reaction is estimated from the rate of oxidation of NADH. The method is rapid, reproducible, and accurate and has been used to estimate diaminopimelate decarboxylase, lysine decarboxylase, and arginine decarboxylase activity, but in principle it can be applied to any decarboxylase which is active in the pH range 6.6 to 7.7. MATERIALS
AND METHODS
L-Arginine hydrochloride, L-arginine decarboxylase (Type IV, from Escherichia coli, 320 units/mg), D,L-diaminopimelic acid, Llysine monohydrochloride, L-lysine decarboxylase (Type VIII, from Bacterium cadaveris, 55 units/mg), pyridoxal 5’-phosphate, and the carbon dioxide diagnostic kit were
0003-2697188 $3.00 Copyright 0 1988 by Academic Ress. Inc. All rights of production in any form reserved.
368
SCRIVEN, WLASICHUK,
from the Sigma Chemical Co. ACS scintillation cocktail, L-[U-‘4C]lysine (300 mCi/ mmol), L-[U-‘*C]arginine (342 mCi/mmol), and [ 1,7-14C]diaminopimelic acid (50 mCi/ mmol) were from Amersham. All other chemicals were of reagent grade. Diaminopimelate decarboxylase was isolated from Bacillus sphaericus IF0 3525 by the method of Asada et al. ( IO) and had a specific activity of 0.56 units/mg protein when assayed using the radiochemical method described below with 1.2 mM diaminopimelate in 50 mM potassium phosphate buffer at pH 6.8 (11). Protein was estimated using the Bio-Rad protein assay, which is based on the method described by Bradford ( 12), using bovine serum albumin as a protein standard. A Beckman DU-8 spectrophotometer thermostatted to 37°C was used for optical measurements. SPECTROPHOTOMETRIC
ASSAY
The carbon dioxide diagnostic kit has two components: a CO, free aqueous diluent and CO2 reagent vials that, upon reconstitution with 6.5 ml of diluent, contain 1.79 mM phosphoenolpyruvate, 0.4 mM NADH, phosphoenolpyruvate carboxylase (0.2 12 units/ml), and malate dehydrogenase ( 1.25 units/ml) in Tris buffer, pH 7.7, at 37°C. This assay solution was used for lysine decarboxylase and diaminopimelate decarboxylase assays. A background rate was estimated by adding substrate (in up to 0.1 ml) to 1 ml of reconstituted solution and monitoring the change in absorbance at 340 nm. Typical background rates are 0.0005 AOD/min. The reaction was then initiated by the addition of enzyme in volumes of up to 50 ~1. The addition of enzyme to the kit components gave no change in absorbance above background, unless substrate was present. For assays at lower pH values, the kit was reconstituted with the COZ free aqueous diluent and the pH adjusted with 0.75 M phosphoric acid ( 1OO- 115 ~1) to give a Tris-phos-
AND PALCIC
phate buffer in the pH range 6.6 to 6.8. In initial trials, the lyophilized kit components were reconstituted with 6.5 ml of 50 mM KH2P04 acidified with phosphoric or sulfuric acid. In this case the buffer component is degassed under vacuum for 10 min prior to use in reconstituting the kit. RADIOCHEMICAL
ASSAY
Diaminopimelate decarboxylase was assayed by monitoring 14C02 evolution from [ 1,7-‘4C]diaminopimelate. Assay mixtures contained 50 mM potassium phosphate buffer or 100 mM Tris buffer at the appropriate pH, substrate, 0.25 PC1 of labeled diaminopimelate, and enzyme in a total volume of 0.86 ml in a scintillation vial. The caps of the vials contained a 1.5 X 1.5~cm piece of filter paper impregnated with 20 ~1 of 1 M hyamine hydroxide solution as a 14C02 trap ping agent. The reaction was initiated by the addition of enzyme and the assay mixture was incubated at 37°C for 20 min with continual shaking. The reaction was terminated by the addition of 0.2 ml of 10% trichloroacetic acid and the vials were shaken for 60 min to ensure that 14C02 evolution was complete. The filter papers were removed and counted in 10 ml of ACS scintillation cocktail on a Beckman LS 180 1 scintillation counter. Lysine decarboxylase was assayed in 100 mM Tris-phosphate buffer, pH 6.7, containing L-lysine, 0.14 rCi labeled L-lysine, and enzyme in a total volume of 1.O ml in a scintillation vial. Following incubation at 37°C for 15 min the reaction was quenched with 0.2 ml of 10% trichloroacetic acid and 14C02 was trapped as described for diaminopimelate decarboxylase. Arginine decarboxylase was assayed in an analogous fashion in 100 mM Tris-phosphate buffer, pH 6.6, containing substrate, 0.13 &i labeled L-arginine, and enzyme in 1.O ml. Incubations were carried out for 10 min at 37”C, quenched, and
A SPECTROPHOTOMETRIC
treated as described for diaminopimelate carboxylase.
369
ASSAY FOR DECARBOXYLASES
de-
RESULTS AND DISCUSSION
The coupled assay was proportional to diaminopimelate decarboxylase concentration, as seen in Fig. 1, where a linear relationship with a correlation coefficient of 0.998 was obtained between AOD/min at 340 nm and enzyme concentration. These absorbance changes correspond to the range of 4.9 to 39 nmol product/min, using a millimolar extinction coefficient of 6.22 mM-’ cm-’ for NADH absorbance at 340 nm. The lower limits that can be quantitated are 1.5 nmol product/min, which corresponds to a change of 0.008 OD/min. The reaction is linear for at least 10 min for concentrations of enzyme giving 0.15 AOD/min or less. At higher concentrations, rapid depletion of NADH causes curvature in 3 to 7 min, but initial rates can still be estimated from tangents to the curves at maximal slopes. The effect of substrate concentration on the initial rate of the diaminopimelate decarboxylase-catalyzed reaction at pH 7.7 is
0.2 -
.G E
75
pg Diaminopimelate
Decarboxylase
FIG. 1. The effect of enzyme concentration on the reaction rate. Diaminopimelate decarboxylase isolated from Bacillus sphaericus was used. The final diaminopimelate concentration was 10 mM, and measurements were carried out in a total volume of 1.15 ml at pH 7.7 and 37’C.
1
OO
5
l/[Diaminopimelate]
I 15
10
(mM-‘)
FIG. 2. The effect of diaminopimelate concentration on the rate of CO* production. The activity was estimated spectrophotometrically at 340 nm in a volume of 1.15 ml at pH 7.7 and 37’C using 25 pg of enzyme.
shown as a reciprocal plot in Fig. 2. These data were analyzed using a computer program based on the statistical method of Wilkinson (13) and gave a V,, of 0.54 f 0.02 pmol/min/mg of protein with a K,,, of 0.67 f 0.06 mM. When the radiochemical method was employed at this pH, values of 0.66 -t 0.06 pmol/min/mg and 1.O + 0.2 mM were obtained for V,,, and Km, respectively. Though the reasons for the discrepancies in these values is unclear, we do not consider these to be major, in view of the great differences in the two methods used. The diaminopimelate decarboxylase assay was also carried out at pH 6.8. For this pH a K,,,of 2.1 f 0.2 mM was obtained for the spectrophotometric assay and 2.3 10.3 mM for the radiochemical method; these values are comparable to 1.8 mM reported by Asada et al. ( 10). The values of V,, are 0.78 f 0.04 and 0.84 * 0.04 pmol/min/mg for the spectrophotometric and radiochemical methods, respectively. Lysine decarboxylase was assayed spectrophotometrically at pH 6.7 and again the dependence of the initial rate upon enzyme concentration was linear with a correlation coefficient of 0.998 (Fig. 3). The effect of lysine concentration on reaction velocity at pH 6.7 is shown as a reciprocal plot in Fig. 4. Analysis of these data using the Wilkinson
370
SCRIVEN, WLASICHUK,
method gave a V,, of 48 + 1pmol/min/mg. Substrate inhibition was observed for lysine concentrations greater than 5 mM (data not plotted). The K, obtained by fitting the initial rate data below this concentration is 0.36 + 0.03 mM. The radiochemical assayat pH 6.7 gave V,, 45 + 7 rmol/min/mg and K,,, 0.42 + 0.04 mM. When arginine decarboxylasewas assayed spectrophotometrically at pH 6.6, a V,,,,,of 75 f 6 pmol/min/mg and a K, of 1.2 + 0.2 mM were obtained. The corresponding values obtained with the radiochemical assay are 71 + 5 rmol/min/mg and 1.Of 0.2 mM for I’,,,, and K,,,, respectively. The pH dependence of the arginine decarboxylasereaction has been studied (14,15) and the relative velocity is optimal from pH 5 to 6, declining sharply beyond this range. However, at pH 6.6 about 25% of the activity remains. This spectrophotometric assay could easily be employed for biosynthetic (16) or plant (3) arginine decarboxylases, which exhibit pH optima of 8 and 7.5, respectively. The overall reproducibility of the assayis good; in initial trials with lysine decarboxylasefor 20 different samplesdone in triplicate the average standard deviation was 4.7%.
AND PALCIC
l/[Lysine]
(mM-‘)
FIG. 4. The effect of lysine concentration on the rate of COz production by lysine decarboxylase at pH 6.7 and 37°C.
This included data at both high and low substrate concentrations. In summary, we report on a rapid, reproducible method for assaying three amino acid decarboxylases:diaminopimelate decarboxylase, lysine decarboxylase,and arginine deearboxylase.We find that reconstitution of a commercial kit for CO2 estimation can be conveniently employed for this purpose, although in principle the kit can be simulated by the individual reagents, which are all commercially available. The activity of these amino acid decarboxylases is reasonably comparable to that obtained using radiochemical methods. ACKNOWLEDGMENTS This work was supported by the Natural Sciences and Engineering Research Council of Canada and the Alberta Heritage Foundation for Medical Research.
REFERENCES
o.cm~,,,_] 0
100
ng Lyslne
zal
Decerboxylase
FIG. 3. The effect of lysine decarboxylase concentration on the reaction rate. The final lysine concentration was 5 mM and measurements were carried out in a volume of 1.15 ml at pH 6.7 and 37°C.
1. Najjar, V. A. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 3, pp. 462-464, Academic Press, New York. 2. Pegg, A. E., and McGill, S. (1979) Biochim. Biophys. Acta 568,416-427. 3. Smith, T. A. (1979) Anal. Biochem. 92, 331-337. 4. Morris, D. R., and Boeker, E. A. (1981) in Methods in Enzymology (Tabor, H. and Tabor, C. W., Eds.), Vol. 94, pp. 125-134, Academic Press, New York.
A SPECTROPHOTOMETRIC
ASSAY FOR DECARBOXYLASES
5. McCaman, M. W., McCaman, R. E., and Lees, G. J. (1972) Anal. Biochem. 45,242-252. 6. Raham, M. D. K., Nagatsu, T., and Kato, T. (1980) J. Chromalogr. 221,265-270. 7. Phan, A. P. H., Ngo, T. T., and Lenhoff, H. M. (1982) Anal. B&hem. 120, 193-197. 8. Rosei, M. A., Avigliano, L., Sabatini, S., and Rigo, A. (1984) Anal. Biochem. 139,73-76. 9. Tonelli, D., Budini, R., Gattavecchia, E., and Girotti, S. (1981) Anal. B&hem. 111, 189-194. 10. Asada, Y., Tanizawa, K., Kawabata, Y., Misono, H., and Soda, K. (198 1) Agric. Biol. Chem. 45, 1513-1514.
371
11. Kelland, J. G., Palcic, M. M., Pickard, M. A., and Vederas, J. C. (1985) Biochemistry 24, 3263-3267.
12. Bradford,
M. M. (1976) Anal. Biochem.
72,
248-254.
13. Wilkinson, G. N. (1960) B&hem. J. 80,324-332. 14. Taylor, E. S., and Gale, E. F. ( 1945) B&hem. J. 39, 52-58.
15. Blethen, S. L., Boeker, E. A., and Snell, E. E. (1968) J. Biol. Chem. 243, 1671-1677. 16. Morris, D. R., and Pardee, A. B. (1966) J. Biol. Chem. 241,3129-3135.