APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1977, p. 604-606 Copyright i 1977 American Society for Microbiology
Vol. 34, No. 5 Printed in U.S.A.
Agar Plate Screening Procedure for Cyclic Adenosine 3',5'Monophosphate and Inhibitors of Cyclic Nucleotide Phosphodiesterase P. J. SOMERS* AND C. E. HIGGENS The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206
Received for publication 23 May 1977
A procedure is described for the semiquantitative measurement of cyclic adenosine 3',5'-monophosphate (cAMP) and detection of inhibitors of cAMP phosphodiesterase by an agar plate test. The assay organism was an adenyl cyclase-deficient mutant derived from Escherichia coli HfrH. In the presence of an acid base indicator, acid production from carbohydrate metabolism was observed as a yellow zone around filter paper disks containing cAMP. Since yellow zone formation reflects the presence of cAMP, a phosphodiesterase inhibitor can be detected indirectly by the presence of a yellow zone on assay plates from a reaction mixture of an inhibitor, phosphodiesterase, and cAMP. Three known cyclic nucleotide phosphodiesterase inhibitors were active against beef brain phosphodiesterase in this system.
Previous reports from other laboratories on the role of cyclic adenosine 3',5'-monophosphate (cAMP) in bacterial metabolism (1, 6-11) provided possible approaches for developing a screening procedure and semiquantitative assay for inhibitors of cyclic nucleotide phosphodiesterase from antimicrobially inactive fermentation broths. A screening system was sought that was relatively rapid and could accommodate large numbers of samples. The stimulation by cAMP of the synthesis of a variety of inducible enzymes, such as,B-galactosidase and galactokinase, became the basis for this procedure. An adenyl cyclase-deficient mutant of Escherichia coli (10) was inoculated into agar containing galactose and an acid-base indicator, bromocresol purple. Filter paper disks containing cAMP were placed on the surface of inoculated plates, and the plates were incubated. Since this organism is unable to metabolize carbohydrates other than glucose in the absence of cAMP (10), acid production from galactose, indicated by a yellow zone, occurs only when galactokinase is induced by the addition of cAMP. Cyclic nucleotide phosphodiesterase will degrade cAMP to 5'-AMP and thus prevent galactose metabolism and acid production. Inhibitors of phosphodiesterase can be detected indirectly in reaction mixtures. Since yellow zone formation indicates the presence of cAMP, a reaction mixture containing cAMP, the enzyme, and a sample of the enzyme inhibitor will produce a yellow zone. The amount of acid production (yellow zone) is proportional to the concentration of phospho-
diesterase inhibitor in the reaction mixture. Adenyl cyclase-deficient mutants derived from E. coli HfrH (10) were cultured overnight at 37°C on agar slants containing 0.69 g of K ,HPOr,, 0.45 g of KH ,PO4, 2.5 g of yeast extract, and 10.0 g of glucose per liter. The cells were harvested, suspended in a solution of 5% lactose and 10% glycerol, and then diluted in the lactose glycerol solution to give an optical density of 0.56 at 580 nm in a Spectronic 20 spectrophotometer. The bacterial concentration was 2 x 107 colony-forming units/ml. The suspensions were frozen and stored in the vapor phase of liquid
nitrogen. Purple agar base (Difco Laboratories, Detroit, Mich.) supplemented with 0.002% bromocresol purple (pH range, 5.2 to 6.8) and 1% galactose (Sigma Chemical Co., St. Louis, Mo.) was seeded with 4 x 105 colony-forming units/ml of agar. The lactose and glycerol concentrations, although 2 and 4 mg/ml of agar, respectively, did not interfere with the plate assay, since they are not metabolized in the absence of cAMP. Each assay plate contained 10 ml. Galactose was the carbohydrate chosen, although lactose, arabinose, mannitol, xylose, glycerol, and maltose also were fernented in the presence of cAMP, but did not produce zones either as large or as sharp as galactose. Filter paper disks (13 mm; Schleicher & Schuell Co., Keene, N.H.) containing cAMP were placed on the agar surface, and the assay plates were incubated overnight at 40°C for maximum yellow zone sizes. Assay plates were also incubated at 30 and 370C, 604
NOTES
VOL. 34, 1977
but 40°C gave the clearest and sharpest zones. Zone size was directly proportional to increments in cAMP concentration over a 20-fold range (Fig. 1). The lowest level of cAMP detectable was 7.2 x 108 mol or 25 ,ug per 13-mm filter paper disk. Each filter paper disk absorbs 100 Beef brain cyclic nucleotide phosphodiesterase was prepared by the method described by Cheung (2). The enzyme solution was diluted in 10 mM phosphate buffer, pH 8.0, to a protein concentration of 1.75 mg/ml. Compounds tested for phosphodiesterase inhibition were dried on 7-mm filter paper disks (Schleicher & Schuell) to remove organic solvents from broth extracts, which might inhibit the enzyme reaction. The paper disks absorb 20 pl of organic solvents and 30 p1 of water when saturated. Dried pads were placed in test tubes containing 100 IlI of enzyme solution (1.75 mg of protein per ml) and incubated in a 30°C water bath. After 1 h of incubation, 100 ,ul of cAMP at 5.8 x 103 M in 10 mM potassium phosphate buffer (pH 8.0) was added, and the tubes were reincubated for another hour. The reaction was terminated by placing the tubes in a steamer for 5 min. Thirteen-millimeter filter paper disks were saturated with the heated reaction mixture and placed on the surface of the assay plate. After overnight incubation, yellow areas around the filter paper disks indicated the presence of cAMP and, consequently, inhibition of the phosphodiesterase. Control reactions of enzyme and substrate at these concentrations converted all cAMP to 5'-AMP, giving no zone formation on assay plates. False positives from fermentation broths that contain glucose or cAMP, or had a low pH, would be identified by the production of a yellow zone around filter paper disks that contained only fermentation broth. Fermentation broths from 1,223 cultures were screened with this procedure. A total of 74 cultures (6%) appeared to produce an inhibitor of phosphodiesterase. Of these 74, 27 were false positives caused by a low pH in the fermentation broth. False positives were obtained with two cultures producing sufficient quantities of cAMP, which was not degraded by the phosphodiesterase enzyme. The remaining 45 cultures are still undergoing evaluation. The three phosphodiesterase inhibitors most often referred to in the literature were active in this system. The miniimal detectable inhibitory concentration was 3 ,Lmol of caffeine, 0.94 ,umol of papaverine, or 4 ,umol of theophylline. Figure 2 shows a dose response of beef brain phosphodiesterase inhibition by papaverine. The yellow zone sizes correspond to the amount of cAMP
605
44- 402 36, 32V; 2824-
20-
10-6 10 -7 cAMP CONCENTRATION MOLES/13 MM FILTER PAPER DISC
FIG. 1. Dose response of yellow zone size to cAMP concentration on assay plates. Assay plates contained 10 ml of purple agar base (Difco) plus 1% galactose and an additional 0.002% bromocresolpurple. The assay organism was an E. coli Hfr mutant deficient in adenyl cyclase. 3331295
27-
s 25-
2321z
1917
* 10- 6
10 - 5
PAPAVERINE MOLES/REACTION MIXTURE
FIG. 2. Dose response ofyellow zone size to papaverine concentrations added to 1.75 mg of beef brain phosphodiesterase per ml and 5.8 x 1-3 M cAMP. Filter paper disks (13 mm) were then placed on agar plates containing 10 ml of purple agar base (Difco) plus 1% galactose and an additional 0.002% bromocresol purple. The assay organism was an E. coli Hfr mutant deficient in adenyl cyclase.
remaining in reaction mixtures. Complete inhibition would be represented by a zone size corresponding to the amount of cAMP added to the reaction mixture. Inhibition of cyclic nucleotide phosphodiesterase could elevate cellular cAMP levels. Compounds that inhibit the mammalian phosphodiesterase enzymes may possess important pharmacological activities such as elevation of fat cell lipolysis (3) and bronchodilation (4). We are grateful to Lester Simon for his assistance in
preparing the beef brain phosphodiesterase and to J. S. Gotts for supplying us with a culture of the adenyl cyclase-deficient E. coli HfrH. LITERATURE CITED 1. Chambers, D. A., and G. Zubay. 1969. The stimulatory effect of cyclic adenosine 3',5'-monophosphate on DNA-
606 2.
3.
4.
5.
6.
APPL. ENVIRON. MICROBIOL.
NOTES
directed synthesis of /i-galactosidase in a cell-free system. Proc. Natl. Acad. Sci. U.S.A. 63:118-122. Cheung, W. Y. 1969. Cyclic 3',5'-nucleotide phosphodiesterase. Preparation of a partially inactive enzyme and its subsequent stimulation by snake venom. Biochim. Biophys. Acta 191:303-315. Dalton, C., F. B. Quinn, C. R. Burghardt, and H. Sheppard. 1970. Investigation of the mechanism of action of the lipolytic agent 4-(3,4-dimethoxybenzyl)-2imidazolidinone (R, 7-2956). J. Pharmacol. Exp. Ther. 173:270-276. Davies, G. E., F. L. Rose, and A. R. Somerville. 1971. New inhibitor of phosphodiesterase with anti-bronchoconstrictor properties. Nature (London) New Biol. 234:50-51. Eron, L, R. Arditle, G. Zubay, S. Connaway, and J. R. Beckwith. 1971. An adenosine 3',5'-cycic monophosphate-binding protein that acts on the transcription process. Proc. Natl. Acad. Sci. U.S.A. 68: 215-218. Pastan, I., and R. Perlman. 1968. The role of the lac promotor locus in the regulation of ,B-galactosidase syn-
7.
8.
9.
10.
11.
thesis by cyclic 3',5'-adenosine monophosphate. Proc. Natl. Acad. Sci. U.S.A. 61:1336-1342. Perlman, R. L., and I. Pastan. 1968. Regulation of I)galactosidase synthesis in Escherichia coli by cyclic AMP. J. Biol. Chem. 243:5420-5427. Perlman, R. L, and I. Pastan. 1969. Pleiotrophic deficiency of carbohydrate utilization in an adenyl cyclase deficient mutant of Escherichia coli. Biochem. Biophys. Res. Commun. 37:151-157. Varmus, H. E., R. L. Perlman, and I. Pastan. 1970. Regulation of lac messenger ribonucleic acid synthesis by cyclic adenosine 3',5'-monophosphate and glucose. J. Biol. Chem. 245: 2259-2267. Yokota, T., and J. Gotts. 1970. Requirement of adenosine 3',5'-cyclic phosphate for flagella formation in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 103:513-516. Zubay, G., D. Schwartz, and J. Beckwith. 1970. Mechanism of activation of catabolite-sensitive genes: a positive control system. Proc. Natl. Acad. Sci. U.S.A. 66: 104-110.
Vol. 34, No. 5 Printed in U.S.A.
Agar Plate Screening Procedure for Cyclic Adenosine 3',5'Monophosphate and Inhibitors of Cyclic Nucleotide Phosphodiesterase P. J. SOMERS* AND C. E. HIGGENS The Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46206
Received for publication 23 May 1977
A procedure is described for the semiquantitative measurement of cyclic adenosine 3',5'-monophosphate (cAMP) and detection of inhibitors of cAMP phosphodiesterase by an agar plate test. The assay organism was an adenyl cyclase-deficient mutant derived from Escherichia coli HfrH. In the presence of an acid base indicator, acid production from carbohydrate metabolism was observed as a yellow zone around filter paper disks containing cAMP. Since yellow zone formation reflects the presence of cAMP, a phosphodiesterase inhibitor can be detected indirectly by the presence of a yellow zone on assay plates from a reaction mixture of an inhibitor, phosphodiesterase, and cAMP. Three known cyclic nucleotide phosphodiesterase inhibitors were active against beef brain phosphodiesterase in this system.
Previous reports from other laboratories on the role of cyclic adenosine 3',5'-monophosphate (cAMP) in bacterial metabolism (1, 6-11) provided possible approaches for developing a screening procedure and semiquantitative assay for inhibitors of cyclic nucleotide phosphodiesterase from antimicrobially inactive fermentation broths. A screening system was sought that was relatively rapid and could accommodate large numbers of samples. The stimulation by cAMP of the synthesis of a variety of inducible enzymes, such as,B-galactosidase and galactokinase, became the basis for this procedure. An adenyl cyclase-deficient mutant of Escherichia coli (10) was inoculated into agar containing galactose and an acid-base indicator, bromocresol purple. Filter paper disks containing cAMP were placed on the surface of inoculated plates, and the plates were incubated. Since this organism is unable to metabolize carbohydrates other than glucose in the absence of cAMP (10), acid production from galactose, indicated by a yellow zone, occurs only when galactokinase is induced by the addition of cAMP. Cyclic nucleotide phosphodiesterase will degrade cAMP to 5'-AMP and thus prevent galactose metabolism and acid production. Inhibitors of phosphodiesterase can be detected indirectly in reaction mixtures. Since yellow zone formation indicates the presence of cAMP, a reaction mixture containing cAMP, the enzyme, and a sample of the enzyme inhibitor will produce a yellow zone. The amount of acid production (yellow zone) is proportional to the concentration of phospho-
diesterase inhibitor in the reaction mixture. Adenyl cyclase-deficient mutants derived from E. coli HfrH (10) were cultured overnight at 37°C on agar slants containing 0.69 g of K ,HPOr,, 0.45 g of KH ,PO4, 2.5 g of yeast extract, and 10.0 g of glucose per liter. The cells were harvested, suspended in a solution of 5% lactose and 10% glycerol, and then diluted in the lactose glycerol solution to give an optical density of 0.56 at 580 nm in a Spectronic 20 spectrophotometer. The bacterial concentration was 2 x 107 colony-forming units/ml. The suspensions were frozen and stored in the vapor phase of liquid
nitrogen. Purple agar base (Difco Laboratories, Detroit, Mich.) supplemented with 0.002% bromocresol purple (pH range, 5.2 to 6.8) and 1% galactose (Sigma Chemical Co., St. Louis, Mo.) was seeded with 4 x 105 colony-forming units/ml of agar. The lactose and glycerol concentrations, although 2 and 4 mg/ml of agar, respectively, did not interfere with the plate assay, since they are not metabolized in the absence of cAMP. Each assay plate contained 10 ml. Galactose was the carbohydrate chosen, although lactose, arabinose, mannitol, xylose, glycerol, and maltose also were fernented in the presence of cAMP, but did not produce zones either as large or as sharp as galactose. Filter paper disks (13 mm; Schleicher & Schuell Co., Keene, N.H.) containing cAMP were placed on the agar surface, and the assay plates were incubated overnight at 40°C for maximum yellow zone sizes. Assay plates were also incubated at 30 and 370C, 604
NOTES
VOL. 34, 1977
but 40°C gave the clearest and sharpest zones. Zone size was directly proportional to increments in cAMP concentration over a 20-fold range (Fig. 1). The lowest level of cAMP detectable was 7.2 x 108 mol or 25 ,ug per 13-mm filter paper disk. Each filter paper disk absorbs 100 Beef brain cyclic nucleotide phosphodiesterase was prepared by the method described by Cheung (2). The enzyme solution was diluted in 10 mM phosphate buffer, pH 8.0, to a protein concentration of 1.75 mg/ml. Compounds tested for phosphodiesterase inhibition were dried on 7-mm filter paper disks (Schleicher & Schuell) to remove organic solvents from broth extracts, which might inhibit the enzyme reaction. The paper disks absorb 20 pl of organic solvents and 30 p1 of water when saturated. Dried pads were placed in test tubes containing 100 IlI of enzyme solution (1.75 mg of protein per ml) and incubated in a 30°C water bath. After 1 h of incubation, 100 ,ul of cAMP at 5.8 x 103 M in 10 mM potassium phosphate buffer (pH 8.0) was added, and the tubes were reincubated for another hour. The reaction was terminated by placing the tubes in a steamer for 5 min. Thirteen-millimeter filter paper disks were saturated with the heated reaction mixture and placed on the surface of the assay plate. After overnight incubation, yellow areas around the filter paper disks indicated the presence of cAMP and, consequently, inhibition of the phosphodiesterase. Control reactions of enzyme and substrate at these concentrations converted all cAMP to 5'-AMP, giving no zone formation on assay plates. False positives from fermentation broths that contain glucose or cAMP, or had a low pH, would be identified by the production of a yellow zone around filter paper disks that contained only fermentation broth. Fermentation broths from 1,223 cultures were screened with this procedure. A total of 74 cultures (6%) appeared to produce an inhibitor of phosphodiesterase. Of these 74, 27 were false positives caused by a low pH in the fermentation broth. False positives were obtained with two cultures producing sufficient quantities of cAMP, which was not degraded by the phosphodiesterase enzyme. The remaining 45 cultures are still undergoing evaluation. The three phosphodiesterase inhibitors most often referred to in the literature were active in this system. The miniimal detectable inhibitory concentration was 3 ,Lmol of caffeine, 0.94 ,umol of papaverine, or 4 ,umol of theophylline. Figure 2 shows a dose response of beef brain phosphodiesterase inhibition by papaverine. The yellow zone sizes correspond to the amount of cAMP
605
44- 402 36, 32V; 2824-
20-
10-6 10 -7 cAMP CONCENTRATION MOLES/13 MM FILTER PAPER DISC
FIG. 1. Dose response of yellow zone size to cAMP concentration on assay plates. Assay plates contained 10 ml of purple agar base (Difco) plus 1% galactose and an additional 0.002% bromocresolpurple. The assay organism was an E. coli Hfr mutant deficient in adenyl cyclase. 3331295
27-
s 25-
2321z
1917
* 10- 6
10 - 5
PAPAVERINE MOLES/REACTION MIXTURE
FIG. 2. Dose response ofyellow zone size to papaverine concentrations added to 1.75 mg of beef brain phosphodiesterase per ml and 5.8 x 1-3 M cAMP. Filter paper disks (13 mm) were then placed on agar plates containing 10 ml of purple agar base (Difco) plus 1% galactose and an additional 0.002% bromocresol purple. The assay organism was an E. coli Hfr mutant deficient in adenyl cyclase.
remaining in reaction mixtures. Complete inhibition would be represented by a zone size corresponding to the amount of cAMP added to the reaction mixture. Inhibition of cyclic nucleotide phosphodiesterase could elevate cellular cAMP levels. Compounds that inhibit the mammalian phosphodiesterase enzymes may possess important pharmacological activities such as elevation of fat cell lipolysis (3) and bronchodilation (4). We are grateful to Lester Simon for his assistance in
preparing the beef brain phosphodiesterase and to J. S. Gotts for supplying us with a culture of the adenyl cyclase-deficient E. coli HfrH. LITERATURE CITED 1. Chambers, D. A., and G. Zubay. 1969. The stimulatory effect of cyclic adenosine 3',5'-monophosphate on DNA-
606 2.
3.
4.
5.
6.
APPL. ENVIRON. MICROBIOL.
NOTES
directed synthesis of /i-galactosidase in a cell-free system. Proc. Natl. Acad. Sci. U.S.A. 63:118-122. Cheung, W. Y. 1969. Cyclic 3',5'-nucleotide phosphodiesterase. Preparation of a partially inactive enzyme and its subsequent stimulation by snake venom. Biochim. Biophys. Acta 191:303-315. Dalton, C., F. B. Quinn, C. R. Burghardt, and H. Sheppard. 1970. Investigation of the mechanism of action of the lipolytic agent 4-(3,4-dimethoxybenzyl)-2imidazolidinone (R, 7-2956). J. Pharmacol. Exp. Ther. 173:270-276. Davies, G. E., F. L. Rose, and A. R. Somerville. 1971. New inhibitor of phosphodiesterase with anti-bronchoconstrictor properties. Nature (London) New Biol. 234:50-51. Eron, L, R. Arditle, G. Zubay, S. Connaway, and J. R. Beckwith. 1971. An adenosine 3',5'-cycic monophosphate-binding protein that acts on the transcription process. Proc. Natl. Acad. Sci. U.S.A. 68: 215-218. Pastan, I., and R. Perlman. 1968. The role of the lac promotor locus in the regulation of ,B-galactosidase syn-
7.
8.
9.
10.
11.
thesis by cyclic 3',5'-adenosine monophosphate. Proc. Natl. Acad. Sci. U.S.A. 61:1336-1342. Perlman, R. L., and I. Pastan. 1968. Regulation of I)galactosidase synthesis in Escherichia coli by cyclic AMP. J. Biol. Chem. 243:5420-5427. Perlman, R. L, and I. Pastan. 1969. Pleiotrophic deficiency of carbohydrate utilization in an adenyl cyclase deficient mutant of Escherichia coli. Biochem. Biophys. Res. Commun. 37:151-157. Varmus, H. E., R. L. Perlman, and I. Pastan. 1970. Regulation of lac messenger ribonucleic acid synthesis by cyclic adenosine 3',5'-monophosphate and glucose. J. Biol. Chem. 245: 2259-2267. Yokota, T., and J. Gotts. 1970. Requirement of adenosine 3',5'-cyclic phosphate for flagella formation in Escherichia coli and Salmonella typhimurium. J. Bacteriol. 103:513-516. Zubay, G., D. Schwartz, and J. Beckwith. 1970. Mechanism of activation of catabolite-sensitive genes: a positive control system. Proc. Natl. Acad. Sci. U.S.A. 66: 104-110.
