Cyclic 3', 5'-Adenosine Monophosphate Phosphodiesterase of ...

Vol. 116, No. 2 Printed in U.S.A.

JOURNAL OF BACTrOLOGY, Nov. 1973, p. 857-866 Copyright 0 1973 American Society for Microbiology

Cyclic 3', 5'-Adenosine Monophosphate Phosphodiesterase of Escherichia coli L. D. NIELSEN, D. MONARD,' AND H. V. RICKENBERG

Division of Research, National Jewish Hospital and Research Center and Department of Biophysics and Genetics, University of Colorado School of Medicine, Denver, Colorado 80206 Received for publication 13 August 1973

The cyclic 3', 5'-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier.

The occurrence of a 3', 5'-adenosine monophosphate phosphodiesterase (c-AMP phosphodiesterase, EC 3.1.4.C) in Escherichia coli was first described by Brana and Chytil in 1966 (3); these authors reported no attempt to purify the enzyme. Okabayashi and Ide purified a 3', 5'-cAMP phosphodiesterase of Serratia marcescens over 1,000-fold (17). Earlier work (13) from this laboratory showed that partially purified cAMP phosphodiesterase of E. coli required for its activity the simultaneous presence of a reduced thiol and, apparently, another protein. We also showed that the c-AMP phosphodiesterase played a regulatory role insofar as mutants of E. coli defective in c-AMP phosphodiesterase were resistant to catabolite repression (14). In this paper we report the partial purification of the c-AMP phosphodiesterase of E. coli and describe certain of its properties, particularly its requirement for iron. MATERIALS AND METHODS Bacterial strains. E. coli K-12 grown on a casein hydrolysate medium to mid-log phase was purchased from Grain Processing Corp. (Muscatine, Iowa) and stored frozen at -20 C. E. coli strain Crookes (ATCC 8739) a prototroph devoid of c-AMP phosphodiesterase activity and resistant to catabolite repression (4) was obtained from the American Type Culture Collection. Strain AB 257, an Hfr type 3 K-12 derivative (2), obtained from E. A. Adelberg, is a methionine auxotroph sensitive to catabolite repression. Strain AB 257 PC-1 is a spontaneous mutant of AB 257 and is resistant to catabolite repression (8); it has not more than one-tenth to one-fifth of the c-AMP phosphodiesterase activity of the parent strain AB 257 when assayed under conditions optimal for the activity of I Present address: Friedrich Miescher Institut, Basel, Switzerland.

857

the parental strain; we have been unable to map the mutation. Strain LA 12G requires thiamine for growth and is also resistant to catabolite repression (11); it was obtained several years ago from W. F. Loomis, Jr. An earlier report from our laboratory (13), claiming that strain LA 12G was defective in c-AMP phosphodiesterase, was in error. Strains AN 120 and AN 180, obtained from G. B. Cox and F. Gibson of Australian National University, Canberra, are F- derivatives of K-12, auxotrophic for arginine and thiamine; strain AN 120 is also defective in its adenosine triphosphatase (5). Strain B was obtained a number of years ago from the laboratory of S. E. Luria. Medium and conditions of growth. Unless otherwise stated, bacteria were grown aerobically at 37 C in a salts medium (15) containing per liter: KH2PO4, 13.6 g; (NH4)2SO,4, 2.0 g; CaCl2, 0.01 g; FeSO4 7H2O, 0.5 mg; and MgSOc-7H20, 0.2 g. The pH of the medium was adjusted to 7 with KOH. Glucose at 10 mM or glycerol at 20 mM served as the source of carbon, and the medium was supplemented with the required amino acids to a final concentration of 0.4 mM. Thiamine, when required, was added to a concentration of 1 &g per ml. Bacterial growth was measured turbidimetrically in the Klett-Summerson colorimeter, and cultures were harvested in mid- or late-log phase. When strain Crookes was cultured for the production of the activator of the c-AMP phosphodiesterase, growth was in New Brunswick fermenters with forced aeration, and the concentration of glucose was 50 mM. Chemicals. Chemical' were of the highest purity commercially available. c-AMP, c-GMP (cyclic 3', 5'guanosine monophosphate), and cyclic 2',3'-adenosine monophosphate, as well as snake venom (Crotalus adamantus) and 5'-nucleotidase, were purchased from Sigma Chemical Co. 3H-c-AMP (20.8 and 28 Ci/mmol) and 3H-c-GMP (0.65 Ci/mmol) were obtained from Schwarz Bioresearch, and 'N-2'-Odibutyryl c-AMP was from Boehringer, Mannheim. Extraction of bacteria. Bacteria were extracted by any one of three equally effective methods; extraction

858

NIELSEN, MONARD, AND RICKENBERG

and all following operations, unless stated otherwise, were carried out at 0 to 4 C. The bacteria were suspended in a volume (milliliters), equal to their wet weight (grams), of 10 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.3 at 0 C; this buffer will be referred to as buffer A. They were then disrupted either by passage through a French press at 5,000 lb/in2 (3.54 x 106 kg/M2), by treatment in a Raytheon 10-kc/s magnetostrictive oscillator for two consecutive 15min periods, or by grinding with an approximately equal weight of levigated alumina. Deoxyribonuclease (to a final concentration of 1 Ag/ml) and Mg'+ (to a final concentration of 1 mM) were added just prior to the disruption of the bacteria. Extracts were centrifuged at 40,000 x g for 20 min, and the centrifugation was repeated if required for the clarification of the extracts. Both c-AMP phosphodiesterase activity and the proteinaceous activator were in this supernatant fluid, which is referred to as crude bacterial extract in the procedure of purification described below. Assay of c-AMP phosphodiesterase. The assay for c-AMP phosphodiesterase represents a modification of the procedure of Thompson and Appleman (19) and of our earlier procedure (13). The principle of the assay is based on the fact that the anion exchange resin, Dowex 2, binds c-AMP but not adenosine. The assay was carried out in two steps. In step I, 'Hlabeled c-AMP is hydrolyzed by the c-AMP phosphodiesterase to 5'-AMP. In step II, an excess of 5'nucleotidase (or snake venom) hydrolyzes the 5'-AMP formed in step I to adenosine. Unhydrolyzed c-AMP, bound to the anion exchange resin, was removed by sedimentation, and the radioactivity in the supernatant fluid, which represents adenosine (and possibly adenine) and hence is a measure of the c-AMP hydrolyzed, was determined. A mixture containing the following was preincubated at 37 C for 3 min in tubes suitable for low-speed centrifugation (concentrations refer to final concentration): Tris-hydrochloride buffer, 50 mM (pH 7, 37 C); dithiothreitol, 2.5 mM; FeCl,, at a concentration indicated for individual experiments; bacterial extract (or partially-purified preparation of enzyme); extract of strain Crookes (or partially-purified preparation of activator), when used. The total volume was brought to 0.15 ml with water, and the reaction was started by the addition of 0.05 ml of 'H-labeled c-AMP (prewarmed to 37 C) to a final concentration of 0.5 mM. Incubation was at 37 C and step I of the reaction was terminated by the immersion of the tubes containing the reaction mixtures in boiling water for 4 min. After cooling to 37 C, 50 pg of 5'-nucleotidase or of snake venom in 0.02 ml of water containing 20 mM MgCl, was added to each tube, and incubation at 37 C was continued for 15 min. A 2.2-ml amount of a 17% (wt/vol) ethanolic slurry of Dowex AG-2X8 (200 to 400 mesh; formate form) was then added, and the mixtures were kept at room temperature for 30 min with occasional shaking. The resin was sedimented by low-speed centrifugation, and 1.0-ml samples of the supernatant fluids were removed to scintillation vials containing 2.0 ml of absolute ethanol and 7.0 ml of scintillation fluid (1, 4-bis-2-(5-phenyloxazolyl)-benzene, 100 mg/liter;

J. BACTERIOL.

2.5-diphenyloxazole, 4 g/liter in toluene). The radioactivity was determined by liquid scintillation counting. The rate of the reaction was followed by measuring the hydrolysis of c-AMP at three time intervals; duplicate samples were assayed for each time point. The activity of a given preparation was calculated from the slope of the line representing the time course of the reaction. One unit of c-AMP phosphodiesterase is defined as the amount of enzyme which hydrolyzes 1 pmol of c-AMP per min under the conditions described. Specific activity is expressed as units per milligram of protein. Protein was determined by the method of Lowry et al. (12); bovine serum albumin served as standard. Iron was measured spectrophotometrically as a bathophenanthroline complex

(7).

Purification of the c-AMP phosphodiesterase. The procedure described here was developed for the purification of the enzyme in relatively high yield. Several other standard procedures were used with variable success. The major problem encountered during the purification of the enzyme was loss of activity due, possibly, to the removal of a proteinaceous effector of the c-AMP phosphodiesterase (or loss of iron from the enzyme) rather than to inherent instability of the enzyme. In this paper we shall refer to this proteinaceous effector, without prejudice as to its mode of action, as "activator"; we shall describe its preparation below. The data on the purification of the c-AMP phosphodiesterase presented in Table 1 were obtained when fractions were assayed in the presence of 200 ;pg of activator. A crude extract of the Crookes strain of E. coli (c-AMP phosphodiesterase negative) could be substituted for the activator if employed at sufficiently high concentrations. Partially purified preparations of the c-AMP phosphodiesterase were completely inactive in the absence of a reduced thiol: Of the thiols tested (Table 2) dithiothreitol was the most effective and was used routinely at a concentration of 2.5 mM. Reduced nicotinamide adenine dinucleotide (NADH) and NADH phosphate (NADPH) also activated the c-AMP phosphodiesterase in crude extracts, but not in purified preparations of the enzyme. The activation was variable-in some experiments the reduced pyridine nucleotides were as effective as dithiothreitol. We employed two different procedures for the removal of the bulk of the protein from crude extracts. Fractionation with acetone was employed in method A, and a combination of precipitation with streptomycin sulfate, heating, and fractionation with acetic acid was in method B. Occasional variability with respect to the concentration of acetone at which the c-AMP phosphodiesterase was precipitated was encountered. Table 1 summarizes the results of typical experiments in which the two methods were employed. Procedural steps, such as the manner of disruption of the bacteria or elution from diethylaminoethyl (DEAE)-cellulose are independent of the use of method A or B. Method A: acetone. Acetone was precooled to -10 C; a volume equal to that of the crude bacterial extract was added slowly with constant stirring to the extract. After 30 min the large precipitate was re-

VOL. 116, 1973

859

c-AMP PHOSPHODIESTERASE OF E. COLI TABLE 1. Purification of E. coli c-AMP phosphodiesterase Total protein (mg)

Step

1. 2. 3. 4.

Crude extractb ........ Precipitation ........ Sephadex G-100 ...... DEAEe ..............

A

B

13,800 1,16 201 55

23,000 4,500' 246 25

Total unitsa (x 10') A

Sp act (U/mg)

B

505 551 448 460 183 78 113 52

B

A

3.7 4.0 9.1 2.06

x x x x

Yield (%)

10' 105 10' 10

2.4 1.02 3.2 2.12

x x x x

A

B

10' 100 100 105 89 83 10' 36 14 10f 22 9.5

Purification A

B

1 11 25 56

1 4.2 10.3 89

One unit = 1 pmol of c-AMP hydrolyzed per min as assayed in the presence of activator. Crude extract: starting material, 250 g (frozen wet weight) of E. coli K-12. Crude extract for method A was prepared in the French press. Crude extract for method B was prepared by treatment of the bacteria in a Raytheon 10-kc/s magnetostrictive oscillator. c Method A: acetone. dMethod B: streptomycin sulfate, heat, acid. Elution of the protein prepared by method A was by the application of successive portions of a buffer containing increasing concentrations of NaCl, whereas in the case of the protein prepared by method B a gradient of NaCl was employed. a "

TABLE 2. Effect of reducing agents on the activity of purified c-AMP phosphodiesterasea Reducing agent

Activity (percent of maximum)

100 Dithiothreitol, 2.5 mM ............... 75 NADPH, 1 mM ...................... 61 NADH, 1 mM ....................... Reduced glutathione, 5 mM .......... 11 Reduced glutathione, 50 mM ......... 46 6 ,B-Mercaptoethanol, 5 mM ........... . 73 fl-Mercaptoethanol, 50 mM Reduced lipoic acid, 5 mM ........... 21 Cysteine, 5 mM ............... 0 None .....................0 a Assays were in the presence of 2 mg of a dialyzed crude extract obtained from strain Crookes, 200 ug of partially purified activator, or 0.01 mM Fe. The stimulation by reducing compounds appeared to be independent of the use of either crude extract, activator, or iron, except in the case of NADPH and NADH. The reduced pyridine nucleotides activated only in the presence of unheated, dialyzed crude extract.

moved by sedimentation and discarded. A second volume of cold acetone, equal to the first, was added slowly with stirring to the supernatant fluid obtained from the first treatment with acetone. After 30 to 60 min, the preparation was centrifuged and the supernatant fluid was discarded. The precipitate was dissolved in a volume of buffer A, corresponding to approximately one-tenth of the volume of the original crude extract. The preparation was then dialyzed exhaustively against buffer A to remove residual acetone. Method B: streptomycin sulfate, heat, acid. Streptomycin sulfate (10% in water) was added to a fmal concentration of 1% to the crude bacterial extract; after several hours in the cold the precipitate

was removed by centrifugation and discarded. The supernatant solution was heated to 60 C in an 80 C water bath and then cooled immediately in an ice bath. The precipitate formed in this treatment was also removed by centrifugation and discarded. Normal acetic acid was added to the supernatant solution to a final concentration of 0.1 N (pH of approximately 3.5), the acidified preparation was kept on ice for 1 h, and the precipitate was collected by centrifugation at 10,000 x g for 15 min. The precipitate was then dissolved in buffer A and the pH was adjusted to 7 with solid NaHCOs. The volume of the preparation at this point was between one-tenth and one-fifth that of the crude extract. The redissolved, dialyzed acetone precipitate or the redissolved acid precipitate, respectively, were then applied in a volume equal to-2 to 3% of the bed volume to a column of Sephadex G-100 equilibrated with buffer A. Good separation of the enzyme was obtained if the total protein (in milligrams) approximated the bed volume of the column (in milliliters). The sample was eluted with buffer A, protein was monitored by ultraviolet (UV) absorbance, and fractions were assayed for c-AMP phosphodiesterase activity (Fig. 1). Fractions showing high activity were pooled and employed for the next step of purification. Anion exchange columns were prepared with Whatman DE-52, equilibrated with buffer A, and degassed with a water aspirator. The c-AMP phosphodiesterase obtained from Sephadex G-100 was applied to the column at a ratio of 1 to 2 mg of protein per ml of bed volume of the exchanger. Protein was eluted by either one of two methods. (i) The column was washed sequentially with portions of buffer A containing 0.1, 0.2, 0.3, and 0.5 M NaCl, respectively. Each portion of buffer corresponded to approximately twice the bed volume of the column. The c-AMP phosphodiesterase was eluted in the 0.3 M NaCl wash; (ii) the column was washed with a volume of buffer A corresponding to twice the bed volume of the column and containing 0.15 M NaCl. Then a gradient of NaCl ranging from 0.15 to 0.3 M NaCl was applied in a volume three

860


J. BACTERIOL.

1.6 1.4

1.2

I E E a) c C4C

1.0 0

0.8

20,000

0.6

C.

E 0.

0.4

10,000

CX 0C

0.2

F-raction

20

10

Vo

140ml

30 J40 Ve 290ml

50 T60

70

Bed Vol.

440ml

FIG. 1. Elution of c-AMP phosphodiesterase (PDE) from Sephadex G-100. A 560-mg amount ofprotein (acid precipitate, method B) in a volume of 7 ml was applied to a Sephadex G-100 column (2.5 by 100 cm) containing a bed volume of 440 ml, and eluted with buffer A, as described in text. Protein (0) was monitored continuously with an ISCO model 610 UVrecorder. c-AMP phosphodiesterase (0) was assayed in the standard assay system using 0.02 ml per fraction and 200 jg of partially purified activator. The void volume (V.) of the column, bed volume, and elution volume (Ve) are indicated. A crude extract of strain Crookes at a concentration times that of the bed volume. An additional bed volume of 0.3 M NaCl was applied at the end of this of approximately 60 mg/ml was incubated for 1 h at gradient to ensure recovery of all the material that 37 C with ribonuclease (RNase) at a final concentracould be eluted at 0.3 M NaCl. Protein was moni- tion of 10 ug/ml. This was followed by dialysis against tored by UV absorbance, and fractions were assayed buffer A. The dialyzed extract was kept for 3 min in a for c-AMP phosphodiesterase activity (Fig. 2). The boiling-water bath, and the precipitate was removed enzyme was eluted as a single peak at an NaCl by centrifugation and discarded. The pH of the concentration of about 0.23 M. Elution procedure (i) supematant fluid was then brought to about 3.5 by has the advantage of rapidity, whereas (ii) yields the addition of 1 N acetic acid, to a final concentraenzyme of relatively greater purity. Fractions ob- tion of 0.1 N acid. The precipitate that formed as a tained from the DE-52 column by either method and result of the acidification was collected by centrifugashowing high c-AMP phosphodiesterase activity were tion at 10,000 x g for 10 min and dissolved in a volume pooled, concentrated by ultrafiltration under N. pres- of buffer A corresponding to approximately one-tenth sure, and dialyzed against buffer A to remove the the voluimae of the original supemate. The pH was NaCl. adjusted to 7 with solid NaHCO,. In some experiPreparation of proteinaceous activator of ments the precipitation with acetic acid was repeated cAMP phosphodiesterase. E. coli strain Crookes for the removal of residual acid-soluble material. The grown on mineral salts medium was employed for the protein in the final acid precipitate corresponded to routine preparation of the activator since such ex- approximately 1% of that of the crude extract. Treattracts were devoid of even a trace of c-AMP phospho- ment of the activator with proteolytic, but not with diesterase activity that might interfere with the assay nucleolytic, enzymes, or prolonged boiling destroyed its activity. Filtration of the activator, prepared as of extraneous c-AMP phosphodiesterase.

VOL. 116, 1973

861

c-AMP PHOSPHODIESTERASE OF E. COLI

6000 1.0

0.8

4000

°0)

0.61.0~~~~~~~~~~~~~ w E 0 X 0.4 E 0 2000 0~a C-J (% cL

0.2

10

20

30

40

50

60

70

80

90

100

110

120

Fraction Number 2. Elution of c-AMP phosphodiesterase (PDE) from DEAE-cellulose. An 80-mg amount of protein (Sephadex G-100 eluate) in a volume of 110 ml was applied to a Whatman DE-52 column (2.5 by 45 cm) containing a bed volume of 180 ml, and eluted using the NaCl gradient method described in the text. Protein and c-AMP phosphodiesterase were monitored as described under Fig. 1. Fractions containing c-AMP phosphodiesterase were pooled and concentrated under N2 pressure in an Amicon ultrafiltration apparatus. FIG.

described, through Sephadex G-100 indicated a molecular weight of approximately 90,000. Activator was prepared routinely from strain Crookes; however, the protein could also be isolated from strain K-12 in the course of the purification of the c-AMP phosphodiesterase. Thus, when method A (see above) was employed, the first acetone precipitate, which had only very little c-AMP phosphodiesterase activity, contained activator. The acetone precipitate was suspended in buffer A and dialyzed against the same buffer, and insoluble material was removed by centrifugation. The supernatant fluid was then submitted to the same procedure as crude extracts of strain Crookes and yielded a preparation of activator identical in its properties with that obtained from Crookes. When method B was employed for the purification of the c-AMP phosphodiesterase, both activator and phosphodiesterase activity were found in the acid precipitate but were separated by chromatography on Sephadex G-100. The peak of activator activity occurred in the region of fraction 25 (Fig. 1), whereas that of the phosphodiesterase was in the region of fraction 36; there was some overlap in the region between the two peaks. Complete separation between the two proteins was achieved by passage of the pooled and concentrated activator obtained from Sephadex G-100 through Sephadex G-200. This procedure, however, entailed considerable loss in the activity of the activator. Similarly, attempts to purify it further by a variety of procedures also led to a severe loss of activity; storage, over a period of weeks, of the protein at either -20 C or at +2 C inactivated it. Our recent experiments suggest that the loss of activity may be due to a dissociation of Fe from the protein. That the activator exerts its effects by acting

as a caffier of Fe is suggested by the data presented in Table 3, which show that Fe associated with the activator is more effective in stimulating the activity of c-AMP phosphodiesterase than is the same concentration of free Fe.

RESULTS

Occurrence of c-AMP phosphodiesterase in different strains of E. coli. Crude extracts of a number of strains of E. coli were assayed for c-AMP phosphodiesterase activity under conditions known to prevent or minimize the dilution effect described below. The data presented in Table 4 show that the activities of a number of strains did not differ greatly. There were two exceptions to this: strain Crookes, when grown on a mineral salts medium, was devoid of c-AMP phosphodiesterase activity, and mutant AB 257PC-1 had only a fraction of the activity found in its wild-type parent, AB 257. Both Crookes and AB 257PC-1 are resistant to catabolite repression (4) and have somewhat higher cellular concentrations of c-AMP than do c-AMP phosphodiesterase-positive strains when grown on any one of several sources of carbon (4). Table 5 shows that the c-AMP phosphodiesterase in crude extracts loses activity upon dilution, but that this loss can be prevented if the enzyme is assayed in the presence of the proteinaceous activator or, less effectively, if Fe or a crude extract of strain Crookes is present

862

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TABLE 3. Comparison of the effects of free Fe and protein-bound Fe on activity of c-AMP phosphodiesterase" Final conon of Protein added o may mixtureprotein-bound Fe Protein addedtoassaymixture mixture m

asinmay

(M ........ Strain Crookes, crude extract, 2000#9 ....... Heated supernatant liquid from dialyzed strain Crookes, crude extract, 200 pg .................... Activator (acid precipitate from heated supernatant liquid), 200 gg ................................... ............. Bovine serum albumin, 200 g ......... ................ None ..................... ............... None ...................... None . ...................................... None . ......................................

of

fre Fena added tof tM)s Fetdrded

M)

itre (

c-AMP phosphodiesterase activity (percent of

Maximum)

8 x 10-

0

66

8 x 10-'

0

61

0 6 x 10-' 0 105 x 10-'

100 18

6x [3 x [3 x l3 x 3x [3 x

10-6

10-1] 10-71 10-7]

10-1]

Verylow 6 34

10-' 51 10-'] aThe c-AMP phosphodiesterase preparation had been purified through the Sephadex G-100 step and contained 0.5 mg of protein/ml and 3 x 10-' M Fe. It had a specific activity of 325,000 U/ml of protein when assayed in the presence of the activator. The final concentration of c-AMP phosphodiesterase in the assay was 10 pg,

and that of Fe contributed by the c-AMP phosphodiesterase was 3 x 10-7 M. b The Fe present in the preparations of activator was assumed to be protein-bound. This was true for the acid precipitate; however, there was probably some free Fe in the crude extract. The values in brackets designate the concentration of Fe in the preparation of the c-AMP phosphodiesterase and may be due to contamination by the reagents.

TABLE 4. c-AMPphosphodiesterase in crude extracts of E. coli Strain

K-12 (commercial) AB 257 AB 257PC-1 LA12-G AN 120 AN 180 B Crookes

Sp act

35,000 30,000 5,000 19,000 20,000 17,000 40,000 None detected

aA 100-pg amount of crude extract protein was assayed in the presence of 2.5 mM dithiothreitol and 2 mg)of Crookes crude extract, as described in Materials and Methods.

during the assay. Preparations of activator differed in their effectiveness in preventing the loss of phosphodiesterase activity; certain preparations of activator were more effective than Fe at a concentration of 20 pg of activator per assay mixture. The concentration of Fe was critical; a concentration of 1 pM Fe was ineffective, whereas a concentration of 100 pM Fe inhibited the phosphodiesterase activity of crude extracts but not of purified c-AMP phosphodiesterase. We have at present no explanation for this observation. A number of proteins, in addition to bovine serum albumin and including pooled fractions obtained from Sephadex G-100 chromatography of extracts of E. coli (other than those containing the activator), were tested for their ability to prevent the dilution effect and were found to be ineffective.

Properties of c-AMP phosphodiesterase: (i) molecular weight. The size of the c-AMP phosphodiesterase was estimated on the basis of filtration through Sephadex G-100. The data presented in Fig. 1 were obtained with a column calibrated by monitoring the elution of RNase (molecular weight 13,500; elution volume [V.] = 340 ml); bovine serum albumin, monomer (molecular weight 67,000, V. = 208 ml); and bovine serum albumin, dimer (molecular weight 134,000, V. = 154 ml). A linear plot of the ratio of elution volume to void volume against the log of the molecular weight (VJIV. versus log molecular weight [where V. is the TABL 5. Effect of dilution on c-AMP phosphodiesterase activity K-12 crude extract" Protein in 0.2-ml assay mixture Percent activity when supplemented with:

(jgg)

ovine K-2 serum etatalbumin extract

100 50 25 10

0 50 75 90

2 mg of 20pjg of actiNothingCroe crude vator

Fe 10-a1 M

~~~extract

48 55 40 0

70 77 77 68

100 78 87 113

100 not tested 103 65

"The specific c-AMP phosphodiesterase activity of the crude extract, when assayed at 100 pg of K-12 protein and in the presence of either 200 pig of activator or 10- M Fe, was 37,000 U/mg and denotes 100% activity. All assays were performed in the presence of 2.5 mM dithiothreitol.

863


VOL. 116, 1973

void volume]) was used to relate the elution volume of the c-AMP phosphodiesterase to its molecular weight. Both the c-AMP phosphodiesterase of crude extracts and the enzyme in its E partially purified form had a molecular weight , 6000 of approximately 28,000. (ii) pH optimum. The data in Fig. 3 show 0. that c-AMP was hydrolyzed optimally at pH 6.8 x 4000 to 7 in our assay system. Phosphate and succinate buffers severely inhibited the activity of c-AMP phosphodiesterase, presumably by the 0 removal of Fe from the assay mixture. 2000 (iii) Dependence of rate of reaction on E concentration of protein. The data presented in Fig. 4 and 5 show that, under our conditions of assay, the rate of the reaction was a rectilin0 2 4 8 16 ear function of both the amount of protein employed and the duration of the assay. The pg protein/0.2ml assay extrapolated zero time values (intercept with FIG. 4. Effect of enzyme concentration activity ordinate) varied considerably with the experi- of c-AMP phosphodiesterase. Enzymeonpurified -

through the DEAE step (Table 1) was assayed as described in text; assay mixtures contained 200 ug of activator. The specific c-AMP phosphodiesterase activity (first portion of curve) was 600,000 U/mg of protein.

a

3

15,000 h

30,000

2

'

-._.

0-

0~

0

I

20,000

E E

4

10,000 F

a) E

10,000

1LI)

E w

0L

0

5,000 -

2

4

6

8

Minutes

II

5.0

6.0

I

I

I

7.0

8.0

9.0

I

pH FIG. 3. Effect of buffers on apparent pH optimum of c-AMPphosphodiesterase (PDE). A 100-Ag amount of a crude extract of strain K-12 was assayed in reaction mixtures containing the following buffers: (A) Tris-maleate, (B) glycylglycine-NaOH, (C) Trishydrochloride, (D) glycine-NaOH. Buffers were 10 mM and contained 2.5 mM dithiothreitol and 2 mM MgCI2. Step 1 of the assay was carried out at the designated pH, and the reaction mixture was then adjusted to pH 8 prior to the addition of the 5'nucleotidase.

FIG. 5. c-AMP phosphodiesterase activity; effects of activator, theophylline, and 5'-AMP. Hydrolysis of c-AMP was measured as described in text; 15 Mg of enzyme purified through the Sephadex G-100 step (Table 1) was employed. Points represent the averages of duplicate determinations. Curve 1, assay in absence of activator; curve 2, assay in presence of 200 Ag of activator; curve 3, assay in presence of 200 Mg of activator and 10 mM theophylline; curve 4, assay in presence of 200 Mg of activator and 0.5 mM 5'-AMP. (The data presented in this figure clearly demonstrate the importance of basing the determination of c-AMP phosphodiesterase activity on the time course of the reaction.)

mental conditions employed such as concentration of enzyme, compounds added to reaction mixture, etc. Rates of reaction were therefore calculated from the slope of the initial, rectilin-

864


ear portion of the line course of the reaction.

representing the time

(iv) Km. Calculation of the data presented in Fig. 6 shows that the c-AMP phosphodiesterase of E. coli has a Km of approximately 0.5 mM c-AMP; in other experiments values ranging from 0.2 mM to 0.5 mM were obtained. The data presented in Fig. 6 were obtained with a preparation that had been purified 100-fold. Purification did not affect the Km. The addition of activator had no effect on the Km of the enzyme but, as expected, increased the Vmax (approximately fivefold in the experiment described in Fig. 6). When c-AMP was employed at concentrations of below 0.01 mM, its rate of hydrolysis followed first-order kinetics in either the presence or absence of activator and irrespective of whether crude extracts or partially purified preparations were employed. (When low substrate concentrations were used, the phosphodiesterase was employed at concentrations as low as 0.2 usg per assay mixture in the presence of Crookes crude extract or activator.) This suggests that, unlike in the case of mammalian c-AMP phosphodiesterases, there are no additional "high affinity, low Ki" c-AMP phosphodiesterases in E. coli. (v) Specificity for 3',5' c-AMP. c-GMP was tested as a substrate at concentrations ranging from 0.001 to 0.5 mM. Neither crude extracts of strain K-12 nor partially purified preparations 60

'aE

50

J. BACTERIOL.

of c-AMP phosphodiesterase hydrolyzed cGMP. c-GMP did not inhibit the hydrolysis of c-AMP, suggesting that it is not a substrate for the E. coli c-AMP phosphodiesterase, at least under the conditions of assay employed. Neither dibutyryl c-AMP at concentrations ranging from 0.1 to 1 mM, nor 2',3' c-AMP at concentrations from 0.1 to 5.0 mM inhibited the hydrolysis of 3', 5' c-AMP by partially purified preparations of c-AMP phosphodiesterase. The occurrence of a 2', 3' c-AMP phosphodiesterase in the periplasm of E. coli has been reported (16). (vi) Effects of divalent cations. Table 6 shows that of the divalent cations tested, Fe was the most effective in stimulating the activity of c-AMP phosphodiesterase. In other experiments (data not shown), the effects of a number of chelating agents were examined. Ethylenediaminetetraacetic acid (EDTA), at 0.1 mM, inhibited c-AMP phosphodiesterase activity by approximately 40%; o-phenanthroline, at 0.5 mM, inhibited by 70%; enterochelin at 0.01 mM (prepared by the method of Langman et al. [10]) inhibited by 55%, whereas ferric-enterochelin at 0.01 mM had no effect on the activity of the phosphodiesterase. Inhibitors of c-AMP phosphodiesterase activity. Theophylline inhibits the activity of mammalian c-AMP phosphodiesterases (9) and has also been reported to inhibit the c-AMP phosphodiesterase of Serratia marcescens (17) and of E. coli (1). However, under our conditions of assay theophylline at concentrations of up to 10 mM (Fig. 5, curve 3) did not inhibit the enzyme. 5'-AMP, the product of the reaction

r-

TABLE 6. Effect of divalent cations on partially purified c-AMP phosphodiesterasea

Without Activator

40 0

x

30

Ion (10-' M final concn)

Activity (% of maximum)

Fe2+ Mn2+ Co2+ Cu2+ Mg2+ Ca2+ Ba2+ Zn2+ Mo2+ None

100 33 22 13 10 10 10 9 10 10

0

E

I.

20

-,

10

t

ctivator

i

1W-w 1

0

2

3

4

5

6

7

8

IS1S Xl0-4 (M -1)

FIG. 6. Apparent Michaelis constant of c-AMP phosphodiesterase. Enzyme, purified through the DEAE step (Table 1), but no longer maximally active, was assayed at 4.7 ;&g per assay mixture in the standard assay system. The enzyme had low, but measurable, activity in the absence of activator. When present, activator was employed at 50 jig per assay mixture; the preparation of activator employed in this experiment activated maximally at this concentration.

a c-AMP phosphodiesterase was purified through DE-52 chromatography and assayed at 7.4 gg/0.2 ml of standard assay mixture containing 2.5 mM dithiothreitol and 0.01 mM of the cation. Maximal specific activity, obtained with 0.01 mM Fe'+, was 213,000 U/mg. Mg2+ was found to have no effect on c-AMP phosphodiesterase activity; however, 2 mM Mge+ was added for the 5'-nucleotidase step in the assay.

VOL. 116, 1973


catalyzed by c-AMP phosphodiesterase, when added at a concentration of 0.5 mM, i.e., equal to that of c-AMP, inhibited the enzyme by approximately 40% (Fig. 5, curve 4). Neither the inhibition by EDTA (see preceding paragraph) nor that by 5'-AMP could be explained by an effect on the 5'-nucleotidase. DISCUSSION A c-AMP phosphodiesterase has been purified approximately 100-fold from a crude extract of E. coli K-12, and the occurrence of the enzyme in a number of other strains of E. coli has been demonstrated. The E. coli c-AMP phosphodiesterase requires a reducing agent as well as Fe for its activity. It appears unlikely, in view of the requirement for a relatively high concentration of reducing agent (Table 2), that its function is solely that' of keeping the iron in its reduced form. Our earlier fmdings (13) indicated that the hydrolysis of c-AMP by extracts of E. coli required the simultaneous presence of the c-AMP phosphodiesterase and a second protein. The data described in the present communication suggest that the role of this second protein, which we term activator, is that of an iron carrier which makes iron available to the c-AMP phosphodiesterase in a form more effective than free iron. The nature of the interaction between the activator and the cAMP phosphodiesterase is not understood at present; it appears unlikely that the activator exerts its function as iron carrier only in relation to the c-AMP phosphodiesterase. The occur, rence of a proteinaceous activator of mammalian c-AMP phosphodiesterase has been reported by Cheung (6), who found that the partially purified mammalian enzyme was largely inactive unless supplemented with the activator. Purified bovine brain c-AMP phosphodiesterase activator (kindly furnished by W. Y. Cheung, St. Jude Children's Research Hospital, Memphis, Tenn.) when added at 0.1 gg/0.2 ml of assay mixture, a concentration known to activate brain phosphodiesterase, did not activate the c-AMP phosphodiesterase of E. coli. Conversely, partially purified activator prepared from strain Crookes did not activate mammalian c-AMP phosphodiesterase (W. Y. Cheung, personal communication). That bacterial c-AMP phosphodiesterases might be metalloproteins was suggested by earlier experiments which showed that the cAMP phosphodiesterases of Bacillus liquefaciens and E. coli were inhibited by reagents that chelated metals (18) and that the c-AMP phosphodiesterase of Serratia marcescens was stimulated twofold by Fe. The

865

simultaneous requirement of the E. coli c-AMP phosphodiesterase for iron and a reducing agent is reminiscent of a similar requirement of pig heart muscle aconitase (20) which catalyzes the reversible dehydration of citrate and isocitrate. The fact that reduced pyridine nucleotides stimulated the c-AMP phosphodiesterase activity effectively in crude extracts (Table 2) suggested the possibility that the redox potential of the cell, as reflected in the ratio of reduced pyridine nucleotides/oxidized pyridine nucleotides, might play a regulatory role by an effect on the activity of the c-AMP phosphodiesterase. Although this possibility seems attractive, trivial explanations for the stimulation of c-AMP phosphodiesterase by reduced pyridine nucleotides cannot be ruled out at present. Presumably, the inability of reduced pyridine nucleotides to activate the partially purified c-AMP phosphodiesterase reflected the loss during purification of (a) component(s) that mediated the reduction (?) of the c-AMP phosphodiesterase by the pyridine nucleotides. That c-AMP phosphodiesterase plays a regulatory role is suggested by the finding (4) that two strains of E. coli, either devoid of (Crookes) or defective (AB 257PC-1) in c-AMP phosphodiesterase, are also resistant to catabolite repression and have generally higher concentration of c-AMP than do strains wild type with respect to catabolite repression. It is clear, however, that the regulatory role of c-AMP phosphodiesterase is a minor one insofar as the source of carbon during growth still affected the cellular concentration of c-AMP in the phosphodiesterase mutants (4). We compared (data not shown) the c-AMP phosphodiesterase activities of several strains of E. coli during growth on a rich medium (tryptone yeast extract [Difcol and glucose) and during growth on salts medium (glucose as source of carbon). We found that the composition of the medium did not affect significantly the specific c-AMP phosphodiesterase activities provided that the assay was carried out under conditions where the dilution effect (Tables 4 and 5) was minimal. However the c-AMP phosphodiesterase activity of crude extracts obtained from cultures grown on the rich medium was much more resistant to loss of activity by dilution than was the activity of cultures grown on the salts medium. Finally, it should be stressed that we have no assurance that the conditions employed for the assay of c-AMP phosphodiesterase in vitro have much bearing on the activity of the enzyme in vivo. It may be calculated that the c-AMP phosphodiesterase activity observed in crude

866

NIELSEN, MONARD, ,AND RICKENBERG

bacterial extracts (Table 4) would bring about the hydrolysis of the cellular c-AMP, if this is assumed to be approximately 0.01 mM (4), in a matter of seconds. It appears likely that in vivo c-AMP phosphodiesterase is active only intermittently; the nature of the control of its activity remains to be explored.

J. BACTERIOL.

6-phosphate. Biochem. Biophys. Res. Commun. 29:303-310.

9. Jost, J.-P., and H. V. Rickenberg. 1971. Cyclic AMP. Annu. Rev. Biochem. 40:741-774. 10. Langman, L., I. G. Young, G. E. Frost, H. Rosenberg, and F. Gibson. 1972. Enterochelin system of iron transport in Escherichia coli: mutations affecting ferric-enterochelin esterase. J. Bacteriol. 112:1142-1149. 11. Loomis, W. F., Jr., and B. Magasanik. 1965. Genetic control of catabolite repression of the lac operon in Escherichia coli. Biochem. Biophys. Res. Commun. ACKNOWLEDGMENTS 20:230-234. We thank Marianne Tecklenburg and Eva Spitz for their 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. excellent technical assistance. Randall. 1951. Protein measurement with the Folin This investigation was supported by Public Health Service phenol reagent. J. Biol. Chem. 193:265-275. grant AM 11046 from the National Institute of Arthritis and 13. Monard, D., J. Janecek, and H. V. Rickenberg. 1969. The Metabolic Diseases and by grant GB 8292 from the National enzymic degradation of 3',5' cyclic AMP in strains of Science Foundation. E. coli sensitive and resistant to catabolite repression. Biochem. Biophys. Res. Commun. 35:584-591. LITERATURE CITED 14. Monard, D., J. Janecek, and H. V. Rickenberg. 1970. Cyclic adenosine monophosphate diesterase activity 1. Aboud, M., and M. Burger. 1971. Cyclic 3,5' adenosine and catabolite repression in E. coli. p. 393-400. In J. monophosphate-phosphodiesterase and the release of Beckwith and D. Zipser (ed.), The lactose operon. Cold catabolite repression of f-galactosidase by exogenous Spring Harbor Laboratory, New York. cyclic 3,5' adenosine monophosphate in Escherichia 15. Monod, J., G. Cohen-Bazire, and M. Cohn. 1951. Sur la coli. Biochem. Biophys. Res. Commun. 43:174-182. biosynthese de la ,-galactosidase (lactase) chez Esche2. Bachmann, B. J. 1972. Pedigrees of some mutant strains richia coli; la specificit6 de l'induction. Biochim. Bioof Escherichia coli K-12. Bacteriol. Rev. 36:525-557. phys. Acta 7:585-599. 3. Brana, H., and F. Chytil. 1965. Splitting of the cyclic 3', 5'-adenosine monophosphate in a cell-free system of 16. Neu, H. C. 1968. The 5'-nucleotidases and cyclic phosphodiesterases (3'-nucleotidases) of the EnterobacEscherichia coli. Folia Microbiol. 11:43-46. teriaceae. J. Bacteriol. 95:1732-1737. 4. Buettner, M. J., E. Spitz, and H. V. Rickenberg. 1973. Cyclic adenosine 3',5'-monophosphate in Escherichia 17. Okabayashi, T., and M. Ide. 1970. Cyclic 3', 5'-nucleotide phosphodiesterase of Serratia marcescens. Biochim. coli. J. Bacteriol. 114:1068-1073. Biophys. Acta 220:116-123. 5. Butlin, J. D., G. B. Cox, and F. Gibson. 1971. Oxidative phosphorylation in Escherichia coli K 12. Mutations 18. Okabayashi, T., and M. Ide. 1970. Effect of dipicolinic acid on bacterial cyclic 3', 5'-nucleotide phosphodiesaffecting magnesium ion- or calcium ion-stimulated terase. Biochim. Biophys. Acta 220:124-126. adenosine triphosphatase. Biochem. J. 124:75-81. 19. Thompson, W. J., and M. M. Appleman. 1971. Multiple 6. Cheung, W. Y. 1970. Cyclic 3', 5'-nucleotide phosphodiescyclic nucleotide phosphodiesterase activities from rat terase. Demonstration of an activator. Biochem. Biobrain. Biochemistry 10:311-316. phys. Res. Commun. 38:533-538. 7. Hawk, P. B. 1965. Hawk's physiological chemistry, p. 20. Villafranca, J. J., and A. S. Mildvan. 1971. The mechanism of aconitase action. II. Magnetic resonance stud1100-1101. 14th ed. McGraw-Hill Book Co., New York. ies of the complexes of enzyme, manganese(II), iron(II), 8. Hsie, A. W., and H. V. Rickenberg. 1967. Catabolite and substrates. J. Biol. Chem. 246:5791-5798. repression in Escherichia coli: the role of glucose-