A rapid batch assay for cyclic AMP phosphodiesterase

ANALYTICAL

BIOCHEMISTRY

84,

551-558 (1978)

A Rapid Batch Assay for Cyclic AMP Phosphodiesterase DAVID Department

R. ZUSMAN

of Bacteriology and Immunology, University Berkeley, California 94720

of California,

Received July 18, 1977: accepted September 13, 1977 A simple new assay for cyclic AMP phosphodiesterase has been devised. The assay uses tritiated cyclic AMP as a substrate and measures the radioactivity present in the reaction product S-AMP. The samples are spotted on PEI-cellulose and then washed batchwise with lo-12 mM LiCl. Under these conditions, over 99.9% of the cyclic AMP is removed, while about 50% of the 5’-AMP product remains. The assay is very rapid and is suitable for hundreds of samples. The assay has been used for following cyclic AMP phosphodiesterase activity from Myxococcus xanthus on native polyacrylamide gels and for screening mutagenized colonies for enzyme activity.

CAMP’ phosphodiesterase is important, since it helps regulate CAMP concentrations by converting CAMP to 5’-AMP (l-4). The enzyme maintains the sensitivity of cells to changing nutritional conditions and environmental stress by providing a mechanism for reducing background levels of CAMP. Several assays have been described for the enzyme (5-7). The most widely used assay is the procedure of Thompson and Appleman (8), which is based on the fact that Dowex 2 binds cAMPbut not adenosine. The assay is usually carried out in two steps. The first step consists of the hydrolysis of 3H-labeled CAMP by the CAMP phosphodiesterase to 5’-AMP. In the following step, an excess of 5’-nucleotidase or snake venom is added to hydrolyze the 5’-AMP to adenosine. The unhydrolyzed CAMP is then bound to the anion exchange resin and removed by sedimentation. The radioactivity in the supernatant fraction, presumably adenosine or adenine, is then used as a measure of the CAMP hydrolyzed. This paper describes a simple, sensitive, and quantitative assay for CAMP phosphodiesterase which contains only one incubation step and which is suitable for large numbers of samples. The assay has been used as part of a continuing study of development in My~~cocc~s xanthus (9). ’ Abbreviations used: CAMP, adenosine 3’:5’-cyclic phosphate; S’-AMP, Y-phosphate; PEI, polyethyleneimine; Tris, tris(hydroxymethyl)aminomethane.

551

adenosine

0003-269717810842-055 1$02.00/O Copyright All rights

0 1978 by Academic Press, Inc. of reproduction in any form reserved.

552

DAVID

R. ZUSMAN

MATERIALS Bacterial

Strains

and Culture

AND METHODS

Conditions

M. xanthus strains DZ-2 (9) and FB (10) were used. Cultures were growr in Casitone yeast extract (CYE) broth or agar which contains 1% Casitone (Difco), 0.5% yeast extract (Difco), and 0.1% MgS04*7Hz0. Cultures were grown at 30 or 35°C with vigorous aeration. Conditions

for Batch

Washing

of Samples

Precoated plastic sheets (20 x 20 cm) of PEI-cellulose (Polygram Cel300 PEI, Brinkman Instruments, Westbury, New York) were ruled into 1 X 3-cm rectangles. Using a pencil and straight edge, a l-mm clear zone was made around each rectangle to minimize intersample contamination during subsequent experiments. Individual rectangles were numbered using light pencil pressure. Samples (10 ~1 of 10 @J/ml) of [8-3H]adenosine 3’:5’-cyclic phosphate (27 Ci/mmol, Amersham/Searle) or 10 ,ul of 5 &i/ml of [2-3H]adenosine 5’-phosphate (20 Ci/mmol, AmersharnSearle) were spotted on the PEI-cellulose rectangles and air dried. The PEI sheets were then washed by floating them upside down in a Pyrex dish containing 250 ml of water or LiCl at concentrations of from 2 to 20 mM. The Pyrex dish was gently agitated (50 rpm) on a gyratory shaker for 15 min. The wash fluid was then removed and replaced with fresh solution. The dish was agitated for an additional 15 min. The PEI-cellulose sheets were then removed from the wash and dried. The rectangles were cut out ofthe plastic sheets and placed in scintillation vials containing a toluene-based scintillation fluid (Omnifluor, New England Nuclear) and the radioactivity was determined. CAMP Phosphodiesterase

Assay

Cells were harvested by centrifugation at 5OOOg for 20 min, washed, and resuspended in 20 mM Tris buffer, pH 7.5. Cells were sonically disrupted and the debris was removed by sedimentation at 5000g for 20 min. The reaction mixture contained 20 mM Tris, pH 7.5, 150 PM CAMP, 0.5 mM mercaptoethanol, 2 mM MgS04, 0.5 &i of [8-3H]cAMP, and enzyme sample in a total of 100 ,ul. (The K, for the M. xanthus CAMP phosphodiesterase is 24 PM.) Incubations were performed for 30 min at 35°C. Aliquots (20 ~1) were removed and spotted on PEI-cellulose rectangles. After air drying, samples were batch washed with 12 mM LiCl and the radioactivity was determined as described above. Controls were run with each washing to determine blank values and percentage [3H]cAMP and 5’-[3H]AMP remaining. In some experiments, the CAMP phosphodiesterase activity present in polyacrylamide gel slices was determined. For these experiments, gel slices were placed in microtiter wells. The slices were then covered with

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553

100 ~1 of reaction mixture. The microtiter plates were then sealed with plastic covers and floated in a water bath at 35°C for 60 min. Aliquots (20.~1) were then spotted on PEI-cellulose rectangles and batch washed as described above. Analysis

of Reaction

Products

Reaction products were spotted on PEI-cellulose sheets and chromatographed as described by Randerath and Randerath (11) using 50 mM LiCl as the solvent. Ultraviolet-absorbing spots corresponding to 5’-AMP, CAMP, adenosine, and inosine were marked. The chromatograms were then cut into 1 x l-cm squares, and the radioactivity was determined. All of the radioactivity was present in these four spots. Polyacrylamide

Gel Electrophoresis

Disc polyacrylamide gels (5% acrylamide) were prepared using the Tris-glycine, riboflavin procedure of Davis (12). Extracts of M. xanthus (100 ~1 of 1 mg/ml of sonicate) were loaded on the gels, and the electrophoresis was carried out at 0°C using about 3 mA per tube. When the tracking dye was near the bottom ofthe tube, the current was turned offand the gels were removed from the tube, frozen with dry ice and sliced into about 70 slices. The slices were placed in microtiter wells and assayed for CAMP phosphodiesterase activity as described above. Autoradiojborographic

Assay

of CAMP Phosphodiesterase

Mutagenized colonies of M. xanthus strain FB were grown at 35°C on CYE agar. Using flat toothpicks, cells were removed and resuspended in 50 ~1 of 4 mM Tris buffer, pH 7.2, in microtiter wells. Care was taken to collect about the same amount of cellular material with each scraping (a density of about 6 x lo8 cells/ml). Lysis of the cells was achieved by adding 20 yl of Brij-58 (1.5%) and 20 ~1 of lysozyme (5 mg/ml) to each well. Suspensions were incubated for about 30 min at 30°C. Extracts were assayed by pipeting 10 ~1 of lysed cells into fresh microtiter plates containing 5 ~1 of incubation mix per microtiter well. The incubation mix contained (per 20 ml): 0.186 ml of [3H]cAMP (1 mCi/ml), 0.20 ml of mercaptoethanol(100 mM), 0.20 ml of MgSO, (0.4 M), 0.5 ml of CAMP (6 mM), and 18.91 ml of H,O. The microtiter plates were covered and incubated for 30 min at 35°C by floating the plates in a water bath. The reaction was terminated by placing the microtiter plates on ice. After incubation, 5 ~1 from each well was spotted on PEI-cellulose squares. The PEI-cellulose plates for these experiments were ruled into 7.5 x 7.5-mm squares. The grooves between squares were cleaned to prevent spillover between samples. The spots were air dried and stored in the cold. The PEI-cellulose plates were washed with 10 mM LiCl

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as described above. After drying, 15 ml of 7% PPO (2,5-diphenyloxazole, New England Nuclear) in diethyl ether was poured evenly over the sheet (13). The PEI plates were air dried and then inverted over R P/S X-Omat X-ray film and stored at -76°C for about 6 days. The film was developed using Kodak liquid X-ray developer (KLX DEV) and Kodak rapid fixer. Dark spots corresponded to 5’-AMP produced during the enzyme incubation. The amount of cellular material present in extracts can be compared by viewing the washed PEI-cellulose plates under ultraviolet light. In this manner, light spots which correspond to colonies with lower cellular material can be eliminated from further consideration. RESULTS

PEI-cellulose is commonly employed for the separation of nucleic acid derivatives using thin-layer chromatographic systems developed by Randerath and Randerath (11). A modification of this approach was used to determine if a multisample batch-washing procedure could be found for a new CAMP phosphodiesterase assay. Since the substrate for the enzyme, CAMP, has less charge than the reaction product, 5’-AMP. different salt concentrations of LiCl were tested for their ability to elute radioactive CAMP or 5’-AMP from commercial PEI-cellulose plastic-backed plates which had been marked into rectangles (see Materials and Methods). Figure 1 shows that 12 mM LiCl was sufficient to elute 99.97% of the C3H]cAMP from the plates, while about 42% of the 5’-[3H]AMP remains. It should be noted that small differences in recovery were found with different batches of commercial PEI plates and to some extent, from experiment to experiment. Therefore, control rectangles containing [3H]cAMP and 5’-[3H]AMP were routinely included in each batch wash and then compared to unwashed samples. In this manner, each experiment was corrected for the 5’-AMP recovery and the r3H]cAMP background.2 A possible problem with this assay might be the underestimation of enzyme activity due to 5’-nucleotidase activity present in crude extracts. The 5’-nucleotidase activity would hydrolyze the 5’-[3H]AMP product to r3H]adenosine which, in turn, may be converted to other nonphosphorylated nucleotides such as [3H]inosine or r3H]adenine. The extent of this problem was investigated by incubating relatively concentrated extracts of M. xanthus, 800 pg of protein/ml, with [3H]cAMP for 50 min at 35°C (Fig. 2). At pH 7.5, 90% of the [3H]cAMP was hydrolyzed by these extracts. Analysis of the reaction products by thin-layer chromatography on PEI-cellulose showed some conversion of 5’-r3H]AMP to r3H]adenosine and r3H]inosine. However, even under these extreme conditions, the * In these experiments, constant sample volumes were used. In experiments where volumes are not constant, standards must be assayed under the same conditions to assure linear recovery.

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r

0.01 N

.5 LiCl Concentration

(mM)

FIG. 1. Elution of [3H]cAMP and 5’-[$H]AMP from PEI-cellulose using a batch-washing procedure. Samples spotted on commercial precoated plastic sheets of PEI-cellulose were batch washed with LiCl solutions. The percentages 5’-AMP remaining (O-O) and CAMP remaining (0-O) are plotted as a function of LiCl concentration.

extent of the underestimation of activity was less than 10%. Thus, in crude extracts of M. xanthus the 5’-nucleotidase activity does not present a significant problem for the assay of CAMP phosphodiesterase. The batch-washing assay is particularly useful for procedures requiring large numbers of samples. For example, the CAMP phosphodiesterase of M. xanrhus was analyzed on native polyacrylamide gels. Figure 3 shows the activity obtained when slices of a 5% gel were incubated with radioactive CAMP (see Materials and Methods). In this experiment, the background radioactivity was about 300 cpm. The peak of activity was reproducible. In gels containing a higher percentage of a&amide, multiple peaks of activity were resolved which may reflect different aggregation states of the enzyme (unpublished data). The batch-washing assay is also useful for the isolation of mutants defective in CAMP phosphodiesterase. Mutagenized colonies of M. xanthus were picked and resuspended in microtiter plate wells where the cells were lysed. [3H]cAMP incubation mix was then added to the lysates

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6.0

6.5

7.0

7.5

8.0

8.5

9.0

PH

FIG. 2. Analysis of reaction products. The fate of [3H]cAMP following incubation with concentrated extracts of M. xanrhus DZ-2 was determined by analysis of the reaction products by thin-layer chromatography on PEI-cellulose. The percentages of total radioactivity present in CAMP (A-A), Y-AMP (O-O), and adenosine plus inosine are plotted as a function of the pH of the incubations. (0 -0)

and the plates were floated in a water bath at 35°C. The reaction mix was then spotted on PEI-cellulose plates, batch washed, and prepared for autoradiofluorography as described under Materials and Methods. A sample autoradiofluorogram is presented in Fig. 4. The dark spots correspond to 5’-[3H]AMP present in each 5 ~1 aliquot of reaction mix. A control lacking cell extract but containing the radioactive incubation mix showed no visible spot (lower left-hand square). Each 20 x 20-cm PEI-cellulose chromatogram was used for the assay of 600 colony lysates. The batch-washing procedure for these plates took only 30 min. In this manner, over 5000 colonies were screened for enzyme activity. Two mutants containing reduced levels of CAMP phosphodiesterase were found. These mutants were characterized by growing them in broth and preparing extracts for analysis. One mutant contained 50% of wild-type activity; the second mutant contained 25%. DISCUSSION

In this paper a rapid batch-washing assay for CAMP phosphodiesterase is described. The assay has several advantages over the standard Thompson and Appleman (8) procedures. The assay is rapid and suitable for the handling of large numbers of samples. For example, hundreds of samples

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Slice Number FIG. 3. Batch-washing assay of CAMP phosphodiesterase following polyacrylamide gel electrophoresis. Crude extracts of M. xanthus DZ-2 were subjected to polyacrylamide gel electrophoresis. The gels were sliced and assayed for phosphodiesterase. The arrow marks the dye front (bromphenol blue).

can be assayed in several hours without the need for time-consuming centrifugation steps. Since the assay measures only the 5’-AMP bound to PEI-cellulose, 3H,0 and other nonphosphorylated contaminants found in commercial preparations of [3H]cAMP do not interfere with the results. The background radioactivity obtained in our laboratory with the Dowex procedure was usually 1000-2000 cpm; in contrast, the background levels obtained here were several hundred counts per minute. The limits of

FIG. 4. Autoradiofluorographic assay of CAMP phosphodiesterase. Semiquantitation of residual 5’-AMP was used to determine the amount of CAMP phosphodiesterase present in extracts of mutagenized colonies of M. xanthus.This sample autoradiofluorogram compares extracts made directly from 47 colonies. Control (no enzyme), column 1 row 6; mutant 1(50% wild-type activity), column 2 row 3; mutant 2 (25% wild-type activity), column 4 row 3.

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DAVID R. ZUSMAN

substrate concentration of this assay method are a function of the specil activity of labeled CAMP. The practical convenient limit of the method similar to the Thompson and Appleman procedure, 10 nM for CAMP. The assay should be applicable to other procaryotic and eucaryot systems. However, contaminating 5’nucleotidase activities may be problem in some extracts. One way to avoid this problem may be to ac another phosphorylated nucleotide to the reaction mix to help spare tl 5’-AMP from hydrolysis. 5’-Nucleotidase activity was not a significai problem in extracts of M. xanthus. An interesting application of the batch-washing assay is its adaptabilit for autoradiography or autoradiofluorography. Semiquantitative data o enzyme activity can be obtained directly by this method without th expensive and time-consuming use of scintillation counting. This is c particular value in the screening of thousands of colonies for mutant lacking CAMP phosphodiesterase activity. This method can also be of us in searching for overproducing mutants. When an interesting spot is founf on an autoradiogram, quantitative data on the amount of 5’-AMP presen can be obtained by simply finding the corresponding square on thl PEI-cellulose sheet, cutting it out, and placing it in a scintillation fluid fo counting. ACKNOWLEDGMENTS This research was supported by Grants GM 20509-04 from the National Institute of Genera Medical Sciences and BMS 75-0923 1 from the National Science Foundation. I would like tc thank Ms. Joanne Uomini for performing the autoradiofluorographic assays for cAMI phosphodiesterase and Dr. Cheryl Morrison for reading the manuscript.

REFERENCES 1. Appleman, M. M., Thompson, W. J., and Russel, T. R. (1973) in Advances in Cyclic Nucleotide Research (Greengard, P., and Robison, G. A., eds.), Vol. 3, Raven Press New York. 2. Monard, D., JaneEek, J., and Rickenberg, H. V. (1969)Biochem. Biophys. Res. Commun 35, 584. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

Chassy, B. M. (1972) Science 175, 1016. Pannbacker, R. G., and Bravard, L. J. (1972) Science 175, 1014. Thompson, W. J., Brooker, G., and Appleman, M. M. (1974)in Methods in Enzymology (Hardman, J. G., and O’Malley, B. W., eds.), Vol. 38, p. 205, Academic Press, New York. Cheung, W. Y. (1974)in Methods in Enzymology (Hardman, J. G., and O’Malley, B. W., eds.), Vol. 38, p. 213, Academic Press, New York. Butcher, R. W. (1974)in Methods in Enzymology (Hardman, J. G., and O’Malley, B. W., eds.), Vol. 38, p. 218, Academic Press, New York. Thompson, W. J., and Appleman, M. M. (1971) Biochemistv 10, 311. Campos, J. M., and Zusman, D. R. (1975) Proc. Nat. Acad. Sci. USA 72, 518. Dworkin, M. (1962) J. Bacterial. 84, 250. Randerath, K., and Randerath, E. (1967) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part B, Academic Press, New York. Davis, B. J. (1964) Ann. N. I’. Acad. Sci. 121, 404-427. Randerath, K. (1970) Anal. Biochem. 34, 188.