ANALYTICAL BIOCHEMISTRY 71, 527-532 (1976)
A Rapid, Sensitive Assay for Adenosine Deaminase' N A N C Y C . G U S T I N AND ROBERT G . K E M P
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53233 Received August 4, 1975; accepted October 23, 1975 A sensitive assay for adenosine deaminase is described that is based upon the separation of [3H]adenosine and [3H]inosine in 0.1 M formic acid on small columns containing the cation-exchange medium, SP-Sephadex. The assay is employed in a study of the inhibition of mouse thymus adenosine deaminase by 2-chloroadenosine.
Interest in the enzyme adenosine deaminase has grown recently with the discovery of a human combined immunodeficiency disease that is accompanied by, and possibly results from, an absence of the deaminase (1,2). Two general methods are usually employed for assay of the enzyme. The easier of the two is based upon the decrease in absorbancy at 265 nm upon conversion of substrate to product (3). This method is not applicable to low substrate concentrations (below 10 tZM) or to solutions that are highly turbid, such as membrane fractions, or to assays in the presence of other metabolites that absorb strongly in the region of 265 nm. A second method overcomes the above mentioned difficulties by employing radioactive substrate and subsequently separating substrate and product by thin-layer or paper chromatography (4) or by paper electrophoresis (5). This method has all of the problems associated with running a large number of assays on thin-layer or paper such as time-consuming operations in applying samples and removing products, development times usually greater than 1 hr, the utilization of a large amount of space for developing tanks, and, occasionally, difficulty in obtaining quantitative recovery of products. In the present communication we present a simplified assay for adenosine deaminase that involves the removal of unreacted radioactive substrate from the product inosine by absorption on a small ion-exchange column. The assay is applied to the determination of kinetic parameters of thymus adenosine deaminase in the presence and absence of an inhibitor, 2-chloroadenosine. METHODS Materials. Adenosine and intestinal adenosine deaminase were purchased from P - L Biochemicals. 2-Chloroadenosine was a product of 1 This work was supported by USPHS Grant CA 16539. 527 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
528
GUSTIN AND KEMP
Sigma Chemical Co. and [3H]H~O, [3H]adenosine, and [~H]inosine were purchased from New England Nuclear Corp. [3H]Adenosine was purified on thin-layer cellulose chromatography using HzO as the developing solvent. Adenosine has an R~ of approximately 0.6. The area corresponding to adenosine was located under uv light and was scraped from the plate. The cellulose powder was stored at -20°C, and the adenosine could be eluted from the powder with water. SP-Sephadex C-25 (Pharmacia) was washed with 0.1 M formic acid until the effluent was near pH 2 as shown by pH indicator paper. Small columns were prepared by adding the Sephadex gel suspension to disposable Pasteur-type pipets containing a small glass wool plug. The columns were packed to a height of 4.5 cm and were washed with an additional two ml of 0.1 M formic acid prior to use. Small plastic funnels may be attached by Tygon tubing to the tops of the columns to facilitate washing. Procedure for incubation. Assays are performed at 37°C in a final volume of 0.5 ml. The incubation mixture contains 50 mM NaTes 2 (pH 7.4), 1 mM EDTA, and the desired concentration of [3H]adenosine (6080,000 cpm). For general assay purposes, a final adenosine concentration of 0.1 mM is used. In practice, the NaTes-EDTA mixture is prepared double strength and then added in a volume of 0.25 ml. Unlabeled adenosine, [3H]adenosine, and HzO are added to give a volume of 0.45 ml, and, after temperature equilibration, the reaction is started by the addition of 0.05 ml of the sample containing adenosine deaminase that has been diluted appropriately in a medium of 100 mM NaTes, 2 mM EDTA, and 0.5 mM DTT, all at pH 7.4. Incubations are terminated by the addition of 50 tzl of 5 M formic acid. This lowers the pH of the reaction mixture to below pH 3.0 which is sufficient to stop the enzymic reaction and at the same time produces a protonated, positively charged form of adenosine. Separation of [3H]inosine from [3H]adenosine. The acidified assay sample is transferred to the SP-Sephadex column and is allowed to enter the bed. The incubation tube is washed with 0.5 ml of 0.1 M fo1Tnic acid and this is applied to the column. The column is further washed with 2.0 ml of 0.1 M formic acid and combined eluates are discarded. A scintillation counting vial is placed under the column, and 3.0 ml of 0.1 M formic acid is applied to the column. More than 90% of the inosine is eluted in this fraction, and it is collected directly into the counting vial. The vials are ready for counting following the addition of 10 ml of Scintiverse (Fisher Scientific) or similar scintillation cocktail with a high aqueous capacity. The radioactivity in a blank (without enzyme) was substracted from the experimental radioactivity. Activity was determined by calculating the nanomoles of adenosine deaminated based upon the specific radioAbbreviations used: Tes, N-tris(hydroxymethyl)methyl-2-aminoethane DTT, dithiothreitol.
sulfonic acid;
ADENOSINE DEAMINASE ASSAY
529
activity in the original reaction mixture. One milliunit of enzyme activity represents the amount of enzyme required to deaminate 1 nmol of adenosine in 1 min under the conditions of the reaction. It is advisable to carry through the assay in duplicate an additional control, particularly if conditions are changed, such as the use of a new batch of ion-exchange gel. This control is the use of commercial, purified adenosine deaminase for a 10- to 20-min incubation in an amount sufficient to convert all of the adenosine to inosine (about 1 unit). The recovered radioactivity in the inosine fraction should approximate the total input of adenosine, and substantial deviation from this value would suggest that the assay or the separation is not functioning properly. Preparation ofthymocyte extracts. Cell suspensions from the thymuses of 8-week-old, male Swiss Albino mice or of 8-week-old, male AKR/J mice were prepared as described by Kemp and Duquesnoy (6). After the cells were washed, they were suspended in 2 ml of an ice-cold solution containing 0.25 M sucrose, 0.1 mM DTT, and 10 mM NaTes (pH 7.4) and sonicated for 8 sec at a setting of four with a Branson sonifier fitted with a microtip (Model W185). The sonicate was centrifuged at 4°C and 10,000g for 20 min, and the supernatant solution was either used immediately or stored frozen before use in experiments described in the following section. RESULTS AND DISCUSSION
Separation of reaction components by ion-exchange chromatography. The pK of the 6-amino group of adenosine is about 3.6 (7) whereas that of
60
I I u.l 4 0
I l
_~ 30
t
_ 20 "6 I0
i
;~
4
6
8 - 1~0 - 1~2 - 1'4
Fraction Number
FIG. i. Behavior of [3H]HzO (D), [aH]inosine ( © ) , and [aM]adenosine (A) on SP-Sephadex C-25. Samples of each compound containing identical counts/min were prepared in 0.5 ml of 50 mM MaTes (pH 7.4) and 1 mM EDTA. 50/xl of 5 M formic acid was added to each of the samples which were subsequently added to identical 4.5-cm columns of SP-Sephadex previously washed with 0.1 M formic acid. The initial effluent was collected (Fraction No. 1). The columns were eluted with 0.1 M formic acid, 0.5-ml fractions were collected, and radioactivity was measured in a scintillation spectrometer. Recoveries of [aH]inosine and [3H]H~O were greater than 95%, whereas less than 0.1% of the total [3H]adenosine was eluted in the first 7 ml.
530
G U S T I N AND K E M P
A
/flO
g l
I
I
I
10 20 30 40 IJg Protein/Assay
0
0
10 20 30 Minutes
FIG. 2. Linearity of adenosine deaminase assay with varying protein (A) and length of time (B). Extracts of the thymus of Swiss Albino mice were prepared as described in the Methods. In (B), the protein content was 1.9/~g/assay.
the sulphopropyl group of SP-Sephadex is 2 or slightly lower. Thus, in 0.1 M formic acid, which has a pH of 2.7, adenosine has a net positive charge, SP-Sephadex has a negative charge, and inosine has no charge. These relationships are the basis for the separation described in the Methods section. A fortuitous weak interaction of inosine with the ionexchange beads permitted the separation of product inosine from tritiated water, a contaminant of most tritiated compounds, and the result was extremely low blanks of the order of 0.1 to 0.2% of the total input in the assay. Figure 1 describes the separation of inosine from [3H]H20 on the SP-Sephadex columns employed in the assay. It can be seen that tritiated HzO alone or that present as a contaminant in inosine or adenosine was eluted before the inosine and was in the 2.5-ml fraction discarded in the assay. The inosine sample placed on the column described in Fig. 1 had been previously evaporated to dryness and then redissolved prior to chromatography. Chromatography of an untreated sample of commercial [SH]inosine gave a "water" peak that was approximately 20% of the total. Similarly, unpurified [all]adenosine gave a substantial peak of 5 to 10% of the total radioactivity in this region. Freshly purified adenosine had about 0.1% tritiated water which increased slowly upon storage. These results indicate the importance of some type of purification of commercial tritiated materials even if the only procedure is that of driving off tritiated water. The collected fraction of inosine overlapped the tritiated water peak by 10% or less (Fig. 1). The blanks would thus be about 1% even if 10% [3H]H20 were present in the adenosine. Complete removal of this overlap could be achieved with slightly longer columns if for some reason no overlap was desirable. The use of [14C] adenosine would also avoid the problems of the contamination in the inosine region. [14C]Adenosine, however, is more costly, and the relatively
ADENOSINE DEAMINASE ASSAY
531
low specific activity of the commercial product makes assays at nanomolar concentrations of adenosine impossible. Characteristics of the assay. Figure 2 describes the protein dependency of the assay (A) and the time course (B) employing 10,000g supernatant fraction of thymus sonicates as the enzyme source. The assay was linear over the 15-fold range of protein concentrations tested and over a time period of 2 to 30 min. Characterization of the product. The product of an incubation of adenosine deaminase with [3H]adenosine was isolated from SP-Sephadex (2.5 to 5.5 ml formic acid eluate) and lyophilized. The lyophilized product was dissolved in a small volume of water, and carrier inosine was added. The solution was divided into two, and each fraction was placed on a separate thin-layer cellulose plate along with parallel samples of several purines and purine ribosides. One plate was developed with water (Re values: inosine, 0.69; adenosine, 0.48; hypoxanthine, 0.50; adenine, 0.28) and the second with t-amyl alcohol:formic acid:water (3:2:l)(Rs values: inosine, 0.43; adenosine, 0.55). The cellulose was removed from the plate in 1-7 cm sections, and the radioactivity in each was determined. In both cases, more than 90% of the total radioactivity cochromatographed with inosine. Inhibition of thymus adenosine deaminase by 2-chloroadenosine. The assay was originally developed to provide a means of examining kinetics of lymphoid cell adenosine deaminase by 2-chloroadenosine. 2-Chloroadenosine has been shown previously to inhibit bovine placental adenosine
1 12
K
4
J~
i
i
I~/Aderlosinex 105 Fro. 3. Inhibition of thymus adenosine deaminase by 2-ch]oroadenosine. Enzyme extracts of the thymus of AKR/J mice were assayed in the presence of varying concentrations of adenosine without inhibitor (O) and with 0. ] mM (r-l) or 1.0 mM (~) 2-chloroadenosine. Additional assays (not shown on the figure) were performed in the presence of 1.0 mM 2-chloroadenosine with lower concentrations of adenosine (15 /~m and 20 /~m). The reciprocal velocity data points were linear with those shown on the figure, but the numbers exceeded the magnitude of the scale shown on the x-axis. Using an expanded scale, the slope of the line through the triangles (A) was estimated from the data points at these lower concentrations of adenosine in addition to those shown.
532
GUSTIN AND KEMP
deaminase (8). In Fig. 3, adenosine concentrations over the range of 10 to 100 /~M were employed. The response of initial velocities to increasing substrate concentrations followed a hyperbolic curve and the double reciprocal plot of data gave a Km of 36 /ZM. Other experiments employing adenosine concentrations as low as 0.5/~M revealed no sigmoid behavior of the velocity vs substrate concentration curve. 2-Chloroadenosine was a competitive inhibitor of the reaction and a KI of approximately 0.14 mM can be calculated from the data of Fig. 3. General c o m m e n t s . The general procedure for separation of the product of a deaminase reaction of SP-Sephadex could presumably be applied for the assay of cytidine, deoxycytidine, and guanosine deaminases. The method described for adenosine deaminase provides a rapid and extremely sensitive assay. Because of the nature of the substrate saturation curve of enzymes that have Michaelis-Menten kinetics, and because the assay measures the fraction of the total substrate employed, the sensitivity is increased as substrate concentration is decreased until the first order region of the substrate saturation curve is reached. This is, of course, the region where substrate concentration is small with respect to Kin. The lower limit of the concentration of substrate that can be used is that dictated by the specific activity of adenosine. We have used substrate concentrations of 0.5/zM and this is by no means a lower limit. The total time required to apply an incubation mixture to a column, wash it, and to collect the sample in a counting vial is about 10-15 rain. In practice we assay a large number of samples at the same time by employing one or more racks to hold the columns. As many as 40 columns have been run simultaneously. In addition to the deaminase assay described here, we routinely employ ion-exchange chromatography on Pasteur pipet columns for the assay of cyclic nucleotide phosphodiesterase (9) and of 5'nucleotidase.
ACKNOWLEDGMENT The authors are indebted to Dr. Lawrence Menahan for helpful discussions during the development of this procedure.
REFERENCES 1. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., and Meuwissen, H. J. (1973) Lancet 2, 836-837. 2. Parkman, R., Gelfand, E. W., Rosen, F. S., Sanderson, A., and Hirschhorn, R. (1975) New Engl. J. Med. 292, 174-179. 3. Kalckar, H. M. (1947)J. Biol. Chem. 167~ 461-475. 4. Cory, J. G., Carreno, A. J., and Rich, M. A., (1971) Enzymologia 40, 232-240. 5. van der Weyden, M. B., Buckley, R. H., and Kelley, W. N. (1974)Biochem. Biophys. Res. Commun. 57, 590-595. 6. Kemp, R. G., and Duquesnoy, R. J. (1975) J. Immunol. 114, 660-664. 7. Alberty, R. A., Smith, R. M., and Bock, R. M. (1951) J. Biol. Chem. 193, 425-442. 8. Maguire, M. H., and Sim, M. K. (1971) Eur. J. Biochem. 23, 22-29. 9. Huang, Y.-C., and Kemp, R. G. (1971), Biochemistry 10, 2278-2283.
A Rapid, Sensitive Assay for Adenosine Deaminase' N A N C Y C . G U S T I N AND ROBERT G . K E M P
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53233 Received August 4, 1975; accepted October 23, 1975 A sensitive assay for adenosine deaminase is described that is based upon the separation of [3H]adenosine and [3H]inosine in 0.1 M formic acid on small columns containing the cation-exchange medium, SP-Sephadex. The assay is employed in a study of the inhibition of mouse thymus adenosine deaminase by 2-chloroadenosine.
Interest in the enzyme adenosine deaminase has grown recently with the discovery of a human combined immunodeficiency disease that is accompanied by, and possibly results from, an absence of the deaminase (1,2). Two general methods are usually employed for assay of the enzyme. The easier of the two is based upon the decrease in absorbancy at 265 nm upon conversion of substrate to product (3). This method is not applicable to low substrate concentrations (below 10 tZM) or to solutions that are highly turbid, such as membrane fractions, or to assays in the presence of other metabolites that absorb strongly in the region of 265 nm. A second method overcomes the above mentioned difficulties by employing radioactive substrate and subsequently separating substrate and product by thin-layer or paper chromatography (4) or by paper electrophoresis (5). This method has all of the problems associated with running a large number of assays on thin-layer or paper such as time-consuming operations in applying samples and removing products, development times usually greater than 1 hr, the utilization of a large amount of space for developing tanks, and, occasionally, difficulty in obtaining quantitative recovery of products. In the present communication we present a simplified assay for adenosine deaminase that involves the removal of unreacted radioactive substrate from the product inosine by absorption on a small ion-exchange column. The assay is applied to the determination of kinetic parameters of thymus adenosine deaminase in the presence and absence of an inhibitor, 2-chloroadenosine. METHODS Materials. Adenosine and intestinal adenosine deaminase were purchased from P - L Biochemicals. 2-Chloroadenosine was a product of 1 This work was supported by USPHS Grant CA 16539. 527 Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
528
GUSTIN AND KEMP
Sigma Chemical Co. and [3H]H~O, [3H]adenosine, and [~H]inosine were purchased from New England Nuclear Corp. [3H]Adenosine was purified on thin-layer cellulose chromatography using HzO as the developing solvent. Adenosine has an R~ of approximately 0.6. The area corresponding to adenosine was located under uv light and was scraped from the plate. The cellulose powder was stored at -20°C, and the adenosine could be eluted from the powder with water. SP-Sephadex C-25 (Pharmacia) was washed with 0.1 M formic acid until the effluent was near pH 2 as shown by pH indicator paper. Small columns were prepared by adding the Sephadex gel suspension to disposable Pasteur-type pipets containing a small glass wool plug. The columns were packed to a height of 4.5 cm and were washed with an additional two ml of 0.1 M formic acid prior to use. Small plastic funnels may be attached by Tygon tubing to the tops of the columns to facilitate washing. Procedure for incubation. Assays are performed at 37°C in a final volume of 0.5 ml. The incubation mixture contains 50 mM NaTes 2 (pH 7.4), 1 mM EDTA, and the desired concentration of [3H]adenosine (6080,000 cpm). For general assay purposes, a final adenosine concentration of 0.1 mM is used. In practice, the NaTes-EDTA mixture is prepared double strength and then added in a volume of 0.25 ml. Unlabeled adenosine, [3H]adenosine, and HzO are added to give a volume of 0.45 ml, and, after temperature equilibration, the reaction is started by the addition of 0.05 ml of the sample containing adenosine deaminase that has been diluted appropriately in a medium of 100 mM NaTes, 2 mM EDTA, and 0.5 mM DTT, all at pH 7.4. Incubations are terminated by the addition of 50 tzl of 5 M formic acid. This lowers the pH of the reaction mixture to below pH 3.0 which is sufficient to stop the enzymic reaction and at the same time produces a protonated, positively charged form of adenosine. Separation of [3H]inosine from [3H]adenosine. The acidified assay sample is transferred to the SP-Sephadex column and is allowed to enter the bed. The incubation tube is washed with 0.5 ml of 0.1 M fo1Tnic acid and this is applied to the column. The column is further washed with 2.0 ml of 0.1 M formic acid and combined eluates are discarded. A scintillation counting vial is placed under the column, and 3.0 ml of 0.1 M formic acid is applied to the column. More than 90% of the inosine is eluted in this fraction, and it is collected directly into the counting vial. The vials are ready for counting following the addition of 10 ml of Scintiverse (Fisher Scientific) or similar scintillation cocktail with a high aqueous capacity. The radioactivity in a blank (without enzyme) was substracted from the experimental radioactivity. Activity was determined by calculating the nanomoles of adenosine deaminated based upon the specific radioAbbreviations used: Tes, N-tris(hydroxymethyl)methyl-2-aminoethane DTT, dithiothreitol.
sulfonic acid;
ADENOSINE DEAMINASE ASSAY
529
activity in the original reaction mixture. One milliunit of enzyme activity represents the amount of enzyme required to deaminate 1 nmol of adenosine in 1 min under the conditions of the reaction. It is advisable to carry through the assay in duplicate an additional control, particularly if conditions are changed, such as the use of a new batch of ion-exchange gel. This control is the use of commercial, purified adenosine deaminase for a 10- to 20-min incubation in an amount sufficient to convert all of the adenosine to inosine (about 1 unit). The recovered radioactivity in the inosine fraction should approximate the total input of adenosine, and substantial deviation from this value would suggest that the assay or the separation is not functioning properly. Preparation ofthymocyte extracts. Cell suspensions from the thymuses of 8-week-old, male Swiss Albino mice or of 8-week-old, male AKR/J mice were prepared as described by Kemp and Duquesnoy (6). After the cells were washed, they were suspended in 2 ml of an ice-cold solution containing 0.25 M sucrose, 0.1 mM DTT, and 10 mM NaTes (pH 7.4) and sonicated for 8 sec at a setting of four with a Branson sonifier fitted with a microtip (Model W185). The sonicate was centrifuged at 4°C and 10,000g for 20 min, and the supernatant solution was either used immediately or stored frozen before use in experiments described in the following section. RESULTS AND DISCUSSION
Separation of reaction components by ion-exchange chromatography. The pK of the 6-amino group of adenosine is about 3.6 (7) whereas that of
60
I I u.l 4 0
I l
_~ 30
t
_ 20 "6 I0
i
;~
4
6
8 - 1~0 - 1~2 - 1'4
Fraction Number
FIG. i. Behavior of [3H]HzO (D), [aH]inosine ( © ) , and [aM]adenosine (A) on SP-Sephadex C-25. Samples of each compound containing identical counts/min were prepared in 0.5 ml of 50 mM MaTes (pH 7.4) and 1 mM EDTA. 50/xl of 5 M formic acid was added to each of the samples which were subsequently added to identical 4.5-cm columns of SP-Sephadex previously washed with 0.1 M formic acid. The initial effluent was collected (Fraction No. 1). The columns were eluted with 0.1 M formic acid, 0.5-ml fractions were collected, and radioactivity was measured in a scintillation spectrometer. Recoveries of [aH]inosine and [3H]H~O were greater than 95%, whereas less than 0.1% of the total [3H]adenosine was eluted in the first 7 ml.
530
G U S T I N AND K E M P
A
/flO
g l
I
I
I
10 20 30 40 IJg Protein/Assay
0
0
10 20 30 Minutes
FIG. 2. Linearity of adenosine deaminase assay with varying protein (A) and length of time (B). Extracts of the thymus of Swiss Albino mice were prepared as described in the Methods. In (B), the protein content was 1.9/~g/assay.
the sulphopropyl group of SP-Sephadex is 2 or slightly lower. Thus, in 0.1 M formic acid, which has a pH of 2.7, adenosine has a net positive charge, SP-Sephadex has a negative charge, and inosine has no charge. These relationships are the basis for the separation described in the Methods section. A fortuitous weak interaction of inosine with the ionexchange beads permitted the separation of product inosine from tritiated water, a contaminant of most tritiated compounds, and the result was extremely low blanks of the order of 0.1 to 0.2% of the total input in the assay. Figure 1 describes the separation of inosine from [3H]H20 on the SP-Sephadex columns employed in the assay. It can be seen that tritiated HzO alone or that present as a contaminant in inosine or adenosine was eluted before the inosine and was in the 2.5-ml fraction discarded in the assay. The inosine sample placed on the column described in Fig. 1 had been previously evaporated to dryness and then redissolved prior to chromatography. Chromatography of an untreated sample of commercial [SH]inosine gave a "water" peak that was approximately 20% of the total. Similarly, unpurified [all]adenosine gave a substantial peak of 5 to 10% of the total radioactivity in this region. Freshly purified adenosine had about 0.1% tritiated water which increased slowly upon storage. These results indicate the importance of some type of purification of commercial tritiated materials even if the only procedure is that of driving off tritiated water. The collected fraction of inosine overlapped the tritiated water peak by 10% or less (Fig. 1). The blanks would thus be about 1% even if 10% [3H]H20 were present in the adenosine. Complete removal of this overlap could be achieved with slightly longer columns if for some reason no overlap was desirable. The use of [14C] adenosine would also avoid the problems of the contamination in the inosine region. [14C]Adenosine, however, is more costly, and the relatively
ADENOSINE DEAMINASE ASSAY
531
low specific activity of the commercial product makes assays at nanomolar concentrations of adenosine impossible. Characteristics of the assay. Figure 2 describes the protein dependency of the assay (A) and the time course (B) employing 10,000g supernatant fraction of thymus sonicates as the enzyme source. The assay was linear over the 15-fold range of protein concentrations tested and over a time period of 2 to 30 min. Characterization of the product. The product of an incubation of adenosine deaminase with [3H]adenosine was isolated from SP-Sephadex (2.5 to 5.5 ml formic acid eluate) and lyophilized. The lyophilized product was dissolved in a small volume of water, and carrier inosine was added. The solution was divided into two, and each fraction was placed on a separate thin-layer cellulose plate along with parallel samples of several purines and purine ribosides. One plate was developed with water (Re values: inosine, 0.69; adenosine, 0.48; hypoxanthine, 0.50; adenine, 0.28) and the second with t-amyl alcohol:formic acid:water (3:2:l)(Rs values: inosine, 0.43; adenosine, 0.55). The cellulose was removed from the plate in 1-7 cm sections, and the radioactivity in each was determined. In both cases, more than 90% of the total radioactivity cochromatographed with inosine. Inhibition of thymus adenosine deaminase by 2-chloroadenosine. The assay was originally developed to provide a means of examining kinetics of lymphoid cell adenosine deaminase by 2-chloroadenosine. 2-Chloroadenosine has been shown previously to inhibit bovine placental adenosine
1 12
K
4
J~
i
i
I~/Aderlosinex 105 Fro. 3. Inhibition of thymus adenosine deaminase by 2-ch]oroadenosine. Enzyme extracts of the thymus of AKR/J mice were assayed in the presence of varying concentrations of adenosine without inhibitor (O) and with 0. ] mM (r-l) or 1.0 mM (~) 2-chloroadenosine. Additional assays (not shown on the figure) were performed in the presence of 1.0 mM 2-chloroadenosine with lower concentrations of adenosine (15 /~m and 20 /~m). The reciprocal velocity data points were linear with those shown on the figure, but the numbers exceeded the magnitude of the scale shown on the x-axis. Using an expanded scale, the slope of the line through the triangles (A) was estimated from the data points at these lower concentrations of adenosine in addition to those shown.
532
GUSTIN AND KEMP
deaminase (8). In Fig. 3, adenosine concentrations over the range of 10 to 100 /~M were employed. The response of initial velocities to increasing substrate concentrations followed a hyperbolic curve and the double reciprocal plot of data gave a Km of 36 /ZM. Other experiments employing adenosine concentrations as low as 0.5/~M revealed no sigmoid behavior of the velocity vs substrate concentration curve. 2-Chloroadenosine was a competitive inhibitor of the reaction and a KI of approximately 0.14 mM can be calculated from the data of Fig. 3. General c o m m e n t s . The general procedure for separation of the product of a deaminase reaction of SP-Sephadex could presumably be applied for the assay of cytidine, deoxycytidine, and guanosine deaminases. The method described for adenosine deaminase provides a rapid and extremely sensitive assay. Because of the nature of the substrate saturation curve of enzymes that have Michaelis-Menten kinetics, and because the assay measures the fraction of the total substrate employed, the sensitivity is increased as substrate concentration is decreased until the first order region of the substrate saturation curve is reached. This is, of course, the region where substrate concentration is small with respect to Kin. The lower limit of the concentration of substrate that can be used is that dictated by the specific activity of adenosine. We have used substrate concentrations of 0.5/zM and this is by no means a lower limit. The total time required to apply an incubation mixture to a column, wash it, and to collect the sample in a counting vial is about 10-15 rain. In practice we assay a large number of samples at the same time by employing one or more racks to hold the columns. As many as 40 columns have been run simultaneously. In addition to the deaminase assay described here, we routinely employ ion-exchange chromatography on Pasteur pipet columns for the assay of cyclic nucleotide phosphodiesterase (9) and of 5'nucleotidase.
ACKNOWLEDGMENT The authors are indebted to Dr. Lawrence Menahan for helpful discussions during the development of this procedure.
REFERENCES 1. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B., and Meuwissen, H. J. (1973) Lancet 2, 836-837. 2. Parkman, R., Gelfand, E. W., Rosen, F. S., Sanderson, A., and Hirschhorn, R. (1975) New Engl. J. Med. 292, 174-179. 3. Kalckar, H. M. (1947)J. Biol. Chem. 167~ 461-475. 4. Cory, J. G., Carreno, A. J., and Rich, M. A., (1971) Enzymologia 40, 232-240. 5. van der Weyden, M. B., Buckley, R. H., and Kelley, W. N. (1974)Biochem. Biophys. Res. Commun. 57, 590-595. 6. Kemp, R. G., and Duquesnoy, R. J. (1975) J. Immunol. 114, 660-664. 7. Alberty, R. A., Smith, R. M., and Bock, R. M. (1951) J. Biol. Chem. 193, 425-442. 8. Maguire, M. H., and Sim, M. K. (1971) Eur. J. Biochem. 23, 22-29. 9. Huang, Y.-C., and Kemp, R. G. (1971), Biochemistry 10, 2278-2283.
