JouRNAL ow BACTRIOLOGY, Sept. 1978, p. 968-975

Vol. 135, No. 3

0021-9193/78/0135-0968$02.00/0 Copyright 0 1978 American Society for Microbiology

Printed in U.S.A.

Induction of Cyclic AMP Phosphodiesterase in Blastocladiella emersonii and Its Relation to Cyclic AMP Metabolism PAUL M. EPSTEINt AND PHILIP M. SILVERMAN* Department of Molecular Biology, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication 21 May 1978

Extracts of vegetative celis of Blastocladiella emersonii contain 5% or less of the cyclic AMP phosphodiesterase activity in zoospore extracts. This difference in activity could be accounted for entirely by an increase in the differential rate of phosphodiesterase synthesis during sporulation, beginning after a lag period of about 60 min and extending for at least an additional 90 min into the 4-h sporulation process. To emine the relation between enzyme synthesis and cyclic nucleotide metabolism, we determined the substrate specificity of phosphodiesterase synthesized during sporulation and partially purified from zoospores. Zoospore extracts contain two components, separable by gel filtration chromatography, with cyclic AMP phosphodiesterase activity. The larger component accounts for 20% of the total activity and the smaller component for 80%. Both components show essentially an absolute substrate specificity for cyclic AMP among several cyclic purine and cyclic pyrimidine nucleotides tested. Nevertheless, we found no change in the total cyclic AMP content of sporulating cells before, during, or after enzyme activity increased. We speculate that some other component of cyclic AMP metabolism or function limits the rate of cyclic AMP hydrolysis in sporulating cells. The aquatic phycomycete Blastocladiella emersonii proliferates in a cell cycle consisting of alternating periods of growth, as a sessile, multinucleate vegetative cell, and quiescence, as a motile, uninucleate zoospore. These two stages of the cycle are separated by the transitional stages of sporulation, when vegetative cells differentiate and cleave into zoospores, and germination, when zoospores differentiate into vegetative cells. Both germination and sporulation are patterned responses elicited by changes in any of a variety of environmental variables; both involve extensive redirection of cellular resources from growth to maintenance or the reverse, depending on the transition (26). Some of the morphological and metabolic events that comprise these responses have been reviewed by Lovett (13). The amounts of cyclic AMP And cyclic GMP at different stages of the cell cycle suggest that both of these compounds contribute to the regulation of Blastocladiella growth and morphogenesis (25). Observations that the levels or activities of several enzymes of cyclic nucleotide metabolism and function are coupled to the cell t Present address: Department of Pharmacology, University of Texas Medical School, Houston, TX 77025.

cycle support this view (16, 23, 33; P. Epstein and P. Silverman, Fed. Proc. 33:1476, 1974). However, the nature of the coupling mechanisms and the relation between the levels of these enzymes and cyclic nucleotide metabolism are not entirely understood. Zoospores contain up to 20 times the cyclic AMP phosphodiesterase activity of the vegetative cells from which they are derived (16; P. Epstein and P. Silverman, Fed. Proc. 33:1476, 1974). Two observations indicate that the mechanisms regulating phosphodiesterase activity operate during the transitional stages of the cell cycle. First, enzyme activity is lost from zoospores early in germination (16), possibly by degradation of the phosphodiesterase protein along with other zoospore proteins no longer required (12). Second, cyclic AMP phosphodiesterase activity remains low in vegetative cells and increases again during sporulation (16; P. Epstein and P. Silverman, Fed. Proc. 33:1476, 1974). This increase was suggested to be the result of de novo synthesis of phosphodiesterase (P. Epstein and P. Silverman, Fed. Proc. 33:1476, 1974), but a study by Maia and Camargo (16) left some doubt as to how much of the difference in enzyme activity between zoo968

VOL. 135, 1978

CYCLIC AMP PHOSPHODIESTERASE IN B. EMERSONII

spores and vegetative cells could be attributed to a real increase in enzyme activity and how much to morphogenetic events at the end of sporulation. In their study Maia and Camargo found the major increase in phosphodiesterase activity to occur during zoospore release (16), when protein synthesis in newly formed zoospores should already have ceased (13). They attributed the increase to preferential incorporation of cyclic AMP phosphodiesterase protein, as compared to total protein, into zoospores (16). The present studies were undertaken to deternine when during sporulation and by what mechanism cyclic AMP phosphodiesterase activity is regulated, and how enzyme activity and cyclic AMP metabolism are interrelated. Our results indicate that the entire increase in cyclic AMP phosphodiesterase activity occurs during an intermediate period of sporulation, well before zoospore release, as part of an extensive, sporulation-specific genetic program. In addition, it appears that another component of cyclic AMP metabolism or function limits cyclic AMP hydrolysis during this period to maintain a constant cyclic AMP level in sporulating cells. In all of our studies we have assumed that Blastocladiella enzymes assayed in vitro as cyclic AMP phosphodiesterases serve exclusively that function in vivo. This assumption appeared justified by experiments indicating that Blastocladiella contains separate enzymes for the hydrolysis of cyclic AMP and cyclic GMP (25, 32). However, the substrate specificities of these enzymes have not been adequately characterized in view of the fact that the in vivo functions of cyclic nucleotide phosphodiesterases have had to be inferred from their in vitro kinetic properties (10, 18). We show here that zoospores contain two components with cyclic AMP phosphodiesterase activity. Both of them exhibit virtually an absolute substrate specificity for cyclic AMP that derives from the inability of the enzymes to bind other cyclic nucleotides at their active sites. Hence, we identify these enzymes as cyclic AMP phosphodiesterases with regard to their cellular function. MATERIALS AND METHODS Preparation of extracts. Cells at different stages of the cell cycle were obtained as previously described (25). The progress and synchrony of sporulation were monitored by the formation of a discharge papillum at the apical surface of sporulating cells or by the release of progeny zoospores (19). Germination was assayed by formation of a germ tube (28). Extracts were prepared by grinding frozen cells (0.6 to 1 g) with a ceramic mortar and pestle. The cells were kept frozen by addition of a small quantity of solid CO2. After the C02 sublimed and the cells began to thaw, they were

969

extracted in 2 volumes of TM buffer, containing 50 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.8) and 5 mM 2-mercaptoethanol. Unbroken cells and debris were removed by centrifugation in the cold for 10 min at 10,000 x g. In some experiments extracts were dialyzed for 4 h in the cold against 1 liter of TM buffer with no loss of activity. The method of extraction is important because we find that sonic disruption yields extracts with much lower cyclic AMP phosphodiesterase activity than does mechanical breakage. We believe this difference in extraction methods accounts for the different levels of activity reported by Maia and Camargo (16) and by Us.

Assays. Cyclic AMP phosphodiesterase activity was measured routinely by a modification of the radiochemical, two-step assay of Brooker et al. (4). Standard reaction mitures (0.2 ml) contained 3 pAmol of tris(hydroxymethyl)aminomethane-hydrochloride buffer (pH 7.8), 1 ,umol of 2-mercaptoethanol, 2 pmol of MgCl2, 0.2 pmol of cyclic [3H]AMP (1,000 cpm/nmol), and extract protein (50 to 350 ug). Incubation was at 35°C for 5 to 6 min, depending on the level of activity. Reactions were terminated by heating in a 90°C bath for 2 min. Ewcherichia coli alkline phoephatase (20 to 50 jg) was then added, and reaction mixtures were incubated for 20 min at 350C. At the end of this time each reaction mixture was added to a scintillation vial containing 0.2 ml of a 50% (vol/vol) suspension of AG1 x 2 (Cl-) anion exchange resin to adsorb and quench remaining cyclic [3H]AMP. Radioactive adenosine in solution was then measured in a liquid scintillation counter. Reactions with no enzyme or with heat-denatured enzyme gave blank values of 7% of input radioactivity. Initial reaction rates were linear with respect to protein concentration at least over the range of 50 to 350 ug, and constant with respect to time until about 70% of the substrate was hydrolyzed. When the reaction reached a yield, 80 to 90% of the radioactivity added as cyclic AMP remained soluble in the presence of the anion exchange resin. One unit of enzyme activity is the amount required to hydrolyze 1 nmol of cyclic AMP per min under the specified conditions. For competition assays, reaction mixtures were as described above, except each contained 1.1 nmol of cyclic [H]AMP (240,000 cpm/nmol), 110 or 1,100 nmol of other (unlabeled) cyclic nucleotides, 90 jg of bovine serum albumin, and 3 ,ug of a zoospore 510 as source of enzyme. Incubation was for 15 min, during which 0.34 nmol of substrate was hydrolyzed in control reactions with no other cyclic nucleotides present. Substrate specificities were determined by paper chromatography of reaction products after prolonged incubation with the enzyme. Standard reaction mixtures contained 32 nmol of cyclic [3H]AMP (2,220 cpm/nmol) or 19 nmol of cyclic [3H]GMP (7,900 cpm/nmol) and 3.1 U of zoospore phosphodiesterase I or 4.6 U of phosphodiesterase II. Phosphodiesterases I and II were prepared from zoospores by gel filtration chromatography as described below, except that the final step was chromatography over Sephadex G75 instead of G100. Reaction mixtures were incubated for 60 min at 350C, heated at 900C for 2 min, and applied

970

EPSTEIN AND SILVERMAN

to Whatman 3MM chromatography paper along with 50 to 100 nmol each of cyclic nucleotide, 5'-nucleotide, and nucleoside standards appropriate to each reaction mixture. These compounds were separated by descending chromatography for 18 h in a solvent system composed of 95% ethanol and 1 M ammonium acetate (7:3, by volume). Each chromatogram was cut into sections for assay in a scintillation counter. All of the radioactivity could be accounted for as one or more of the standard compounds lised above. For cyclic AMP asays, frozen cells (0.15 to 0.2 g) were extracted in 1 ml of cold, 1 N acetic acid for 60 min, as previously described (25). Acid-soluble fractions were lyophilized to dryness, the residues were dissolved in 0.1 ml of 50 mM sodium acetate buffer (pH 4.0), and cyclic AMP was measured at three levels over a fourfold concentration range as described by Gilman (8). Protein was measired as described by Lowry et al. (14) with bovine serum albumin as the standard. DNA was described by Giles and Myers (7) with calf thymus DNA as the standard. Gel filtration chromatography. Zoospores were obtained by spontaneous sporulation on nutrient agar, filtered through Sargent 500 paper to remove vegetative cells and debris, concentrated by centrifugation at 2,500 x g for 5 min, and frozen at -70°C. The frozen cells (7.6 g) were ground to a fine powder in the presence of solid C02 and 10 unol of phenylmethylsulfonylfluoride. As the cells thawed, they were extracted with 15 ml of buffer A, which contains 50 mM

tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.8), 5 mM 2-mercaptoethanol, and 10 mM MgC12. The crude extract was fractionated by centrifugation in the cold at 10,000 x g for 10 min. The supernatant fluid was removed (S10; 21 ml, 4,431 total cyclic AMP phosphodiesterase units; 14.8 U/mg of protein). The extract was then subjected to centrifugation in the cold at 105,000 x g for 150 min. The supernatant fluid was again removed (S10; 20 ml, 3,820 total units; 26.5 U/mg of protein). Ammonium sulfate (2.68 g) was slowly added to the 5105 fraction with stirring. The precipitate was removed by centrifugation at 30,000 x g for 20 min. Additional ammonium sulfate (2.92 g) was added to the supernatant fluid. The precipitate was collected by centifugation and dissolved in 3.5 ml of buffer A containing 5% (vol/vol) glycerol (25 to 50% ammonium sulfate fraction; 4 mL 3,530 U, 54.8 U/mg). The entire ammonium sulfate fraction was washed into a Sephadex G100 column (38 by 2.8 cm) previously equilibrated with buffer A. The column was eluted with buffer A at a flow rate of 19 ml/h into 3.3-ml fractions. An aliquot (20 pl) of each fraction was used to assay cyclic AMP phosphodiesterase activity. Recovery of activity from the column corresponded to 3,971 U. Peak fractions of phosphodiesterase I (see Fig. 2) had a specific activity of 64 U/mg, and of phosphodiesterase I, 350 U/mg. Materials. Unless otherwise specified, materiaLs were as previously described (25, 27) or obtained from standard commercial sources. Radioactive cyclic nucleotides were purified before use by alumina and ionexchange chromatography (17). Bacterial alkaline phosphatase was obtained as an ammonium sulfate

J. BACTrERIOL. suspension from Worthington Biochemicals (code BAPC). Before use the protein was collected by centrifgation, dissolved in 50 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5) at a concentration of 10 mg/ml, and dialyzed against the same buffer for 4 h. AG1 x 2 (200 to 400 mesh) (C-) was obtained from Bio-Rad Laboratories and washed before use essentially as described by Boudreau and Drummond (2), except that overnight storage in 0.1 M HCl was omitted.

RESULTS

Cyclic AMP phosphodiesterase synthesis in sporulating cells. Extracts prepared from vegetative cella grown for 5 h (6 to 8 nuclei per cell) contain about 1 U of cyclic AMP phosphodiesterase activity per mg of protein, whereas zoospore extracts contain 15 to 20 U/mg for cells obtained by spontaneous sporulation on nutrient agar, or 10 to 15 U/mg for cells obtained by induced sporulation in liquid medium (Table 1). As shown below, the different activities of zoospore and vegetative cell extracts can be attributed entirely to phosphodiesterase synthesized during sporulation, beginning after a lag period of about 60 min and extending over at least a 90-min interval, when the specific activity of the sporulating cells reaches that of the zoospores derived from them (Table 1). TABLE 1. Cyclic AP phosphidiesterase activity during the cell cycle of B. emersonji Cyclic AMP phosphodiProtein (mg Source of extracta

per i10

esterase

(U per le

cells)

(U/mg)

Zoospores Germlings Vegetative cells Early sporulating

7.9 4.6 32.5 26.0

18.2 3.5 0.7 1.7

144 16 23 44

cells Late sporulating cells Progeny zoospores

22.4

9.8

219

cells)

9.4

Zoospores were obtained by spontaneous sporu-

lation on nutrient agar (27). One portion of these cells was harvested by centrifugation, and the remainder was inoculated into synthetic growth medium. Germlings were harvested after 45 min at 270C. Ninety-five percent of these cells had a prominent germ tube. Vegetative cells were harvested 4.5 h later. Sporulating cells and zoospores were obtained in another experiment from vegetative cells grown as described above. The growth medium was exchanged for sporulation solution (28), and samples were harvested 60 min (early sporulating cells) and 150 min (late sporulating cells) later. After 240 min, progeny zoospores (1.2 x 106 cells per ml) were filtered over Sargent no. 500 paper and harvested by centrifugation. Preparation of extracts and assays were as described in the text.

VOL. 135, 1978

CYCLIC AMP PHOSPHODIESTERASE IN B. EMERSONII

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971

thesis is required over an extended interval even within single cells, and are compatible with de novo synthesis either of enzyme protein or of an activator protein that interacts with the enzyme rapidly and stoichiometrically. As shown below, the molecular weight of the kinetically predominant zoospore phosphodiesterase appears too small (Mr = 45,000) to accommodate both the enzyme protein and an activator macromolecule. It is therefore more likely that the increase in enzyme activity is the result of de novo enzyme

synthesis.

Our results also suggest that the enzyme has a low turnover rate in sporulating cells, since the

amount of activity did not appreciably decrease for at least 60 min after addition of cycloheximide to inhibit further synthesis (Fig. 1). The enzyme must also be stable in zoospores, since these cells have a high level of activity and are TIME AFTER STARVATION (min) essentially quiescent with respect to new protein FIG. 1. Kinetics of cyclic AMP phosphodiesterase synthesis. In contrast, enzyme activity decreases synthesis during sporulation. Cells at different stages about 5- to 10-fold during the first 20 min of of sporulation were harvested, extracted, and assayed germination (Table 1; ref. 16), suggesting actifor enzyme activity as described in the text. At 100 vation a of mechanism for protein degradation min after starvation the culture was split, and one half was incubated further with cycloheximide (5 at or near the onset of germination. Lodi and pg/mi). In this experiment the progress and syn- Sonnebom have reported that the rate of bulk chrony of sporulation were monitored by formation protein degradation increases fourfold at the of a discharge papillum on the apical surface of onset of germination (12). Both the increase in sporulating cells. (0) Enzyme activity; (0) enzyme degradation rate (12) and the loss of phosphoactivity in cells treated with cycloheximide; (A) pro- diesterase activity (16) are insensitive to cycloportion of control cells with a discharge papillum. heximide, which blocks late germination events (13, 27, 29). A further decrease in specific activity The lag period preceding the increase in phos- during growth is attributable to accumulation of phodiesterase activity (Fig. 1) corresponds to the protein rather than to any further change in the time required for protein synthesis in sporulat- amount of enzyme in the cells, which remains ing celLs to recover after starvation (13). More- constant at about 20 U per 109 cells for at least over, inhibitors of protein and RNA synthesis the first 5 h of growth (Table 1). added at the onset of sporulation completely Substrate specificity of Blastocladiella block any increase in phosphodiesterase activity cyclic AMP phosphodiesterase. Cyclic AMP (P. Epstein and P. Silverman, Fed. Proc. phosphodiesterase was partially purified from 33:1476, 1974). These observations suggest that zoospore extracts by differential centrifugation, protein synthesis is required for the increase in ammonium sulfate fractionation, and gel filtraphosphodiesterase activity. To demonstrate this tion chromatography (Fig. 2). The ammonium requirement, cycloheximide added after enzyme sulfate fraction contained two active compoactivity began to accumulate was shown to block nents separable by Sephadex G100 chromatogany further accumulation completely and imraphy (Fig. 2A). The larger phosphodiesterase I mediately (Fig. 1). Therefore, the increase in accounted for 20% of the activity applied to the phosphodiesterase activity in the cell population column and the smaller phosphodiesterase II for requires continuous protein synthesis over an 80%. Rechromatography of isolated fractions extended interval. This interval cannot be ex- showed that each of the separated enzymes was plained by population asynchrony, which can be essentially free of the other (Fig. 2B, C). Phosestimated from the fraction of cells with a dis- phodiesterase I eluted from the column slightly charge papillum as a function of time (19). Es- ahead of bovine serum albumin (Mr = 68,000) sentially all of the cells form a discharge papil- added to the sample as a molecular weight lum between 60 and 120 min after starvation, marker, and phosphodiesterase II eluted at the whereas enzyme accumulation, which begins at same position as ovalbumin (Mr = 45,000). about the same time, extends at least to 160 min We detect phosphodiesterase I only when the (Fig. 1). These results imply that protein syn- ammonium sulfate fraction is chromatographed c

972

EPSTEIN AND SILVERMAN

J. BACTERIOL.

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FRACTION NUMBER FIG. 2. Gel filtration chromatography of zoospore cyclic AMP phosphodiesterase. (A) A 25 to 50% ammonium sulfate fraction, prepared as described in the text. (B) Rechromatography of fractions 34 and 35 containing 703 phosphodiesterase units and 3.1 mg ofprotein. Bovine serum albumin (12 mg) and ovalbumin (15 mg) uwre added as molecular weight markers. Recovery of activity from the column corresponded to 289 U. (C) Rechromatography of fractions 25 and 26, containing 5.9 mg of protein and 312 phosphodiesterase units. Before chromatography, bovine serum albumin (20 mg) was added as a molecular weight marker. Recovery of activity fiom the column corresponded to 198 U. In all cases, percentage of transmittance (280 nm) of the eluate was continuously monitored. (O) Enzyme activity; (-) percentage of transmittance (80 nm).

immediately, and isolated phosphodiesterase I is unstable during storage at 40C and to cycles of freezing and thawing. Loss of this component would explain the observation of Vale et al. (32) that 20% of the cyclic AMP phosphodiesterase activity in a zoospore 105,000 x g supernatant fraction was lost within 24 h at 40C, whereas the remaining 80% of the activity was stable for up to 8 days. Zoospore cyclic AMP phosphodiesterases I and II, separated from each other as described above, were highly specific for cyclic AMP as substrate, as determined by paper chromatography of reaction mixtures containing enzyme and either cyclic [3H]AMP or cyclic [9H]GMP as substrate (see Materials and Methods for experimental details). Even after prolonged incubation, such that more than 99% of the cyclic

AMP was hydrolyzed, more than 99% of the cyclic GMP was recovered. We estimate that the activity of zoospore phosphodiesterase I with cyclic AMP as substrate exceeds by at least 300fold its activity with cyclic GMP. This is a minimal estimate, because a small amount of cyclic GMP hydrolysis that we observed may have been catalyzed by traces of a different enzyme specific for that nucleotide (32). Phosphodiesterase II was at least 1,000 times more active with cyclic AMP than with cyclic GMP. The substrate specificity of zoospore cyclic AMP phosphodiesterases derives at least in part from their low affinity for other cyclic nucleotides, as shown by competition experiments (see Materials and Methods for experimental details). At 100-fold molar excess over cyclic [3H]AMP substrate, present at a concentration

CYCLIC AMP PHOSPHODIESTERASE IN B. EMERSONII

VOL. 135, 1978

of 5.5 uM, comparable to the apparent Km of the kinetically predominant phosphodiesterase II (32; P. Epstein, Ph.D. Thesis, Albert Einstein College of Medicine, New York, 1975), cyclic GMP, cyclic CMP, cyclic UMP, and cyclic IMP had little effect on the rate of cyclic AMP hydrolysis. Even at 1,000-fold molar excess, only cyclic GMP inhibited cyclic AMP hydrolysis by as much as 50%. We have carried out the same experiments with enzyme isolated from vegetative cells with the same results. Since cyclic GMP is never present in Blastocladiella in significant excess over cyclic AMP (25), we conclude that Blastocladiella enzymes assayed in vitro as cyclic AMP phosphodiesterases serve only that function in vivo. Effect of cyclic AMP phosphodiesterase induction on cyclic AMP metabolism. Bourne et aL (3) have shown that induction of cyclic nucleotide phosphodiesterase in a line of cultured lymphoma cells is a response to elevated cyclic AMP content in the presence of a functional cyclic AMP-dependent protein kinase. This, or a similar mechanism, may also operate in other mammalian cell lines (6,15,21); however, it is not known to operate in cells of lower eucaryotes. A characteristic feature of the mechanism is that phosphodiesterase induction is preceded by an increase in total cellular cyclic AMP content (3, 6, 15, 21). To determine whether or not this mechanism might operate during sporulation,

973

we compared the cyclic AMP and cyclic AMP phosphodiesterase contents of cells harvested at different stages of sporulation. We have shown that the cyclic AMP content of sporulating cells is constant through the first 2.5 h of sporulation (25). Nevertheless, to make a comparison completely valid, we measured both parameters in samples of the same cell population. As shown (Fig. 3), the cyclic AMP content of sporulating cells, about 1.1 pmol/Ag of DNA, was unaltered over the interval when cyclic AMP phosphodiesterase activity increased. This experiment, along with the previous results (25), rules out a regulatory mechanism in which cyclic AMP phosphodiesterase is synthesized during sporulation in response to an elevated cyclic AMP content. A striking implication of the experiment illustrated in Fig. 3, along with the fact that cyclic AMP tums over during sporulation (25), is that the relative rates of cyclic AMP synthesis and loss remain constant over an interval when phosphodiesterase activity increases more than 10fold. As will be discussed, this could occur because of conditions which do not exist in vitro and which limit the rate of cyclic AMP hydrolysis in vivo; an example would be if substantial amounts of cellular cyclic AMP were not available for hydrolysis. Finally, this experiment shows clearly that phosphodiesterase activity increases well before zoospore release and cannot therefore be ac-

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TIME AFTER STARVATION (min) FIG. 3. Comparison of cyclic AMP and cyclic AMP phosphodiesterase contents of sporulating cells. Sporulating ceUs were obtained and analyzed as described in the text. Open and filed symbols represent the results of two experiments. Circles, Cyclic AMP content; triangles, cyclic AMP phosphodiesterase activity; crosses, zoospore release.

974

EPSTEIN AND SILVERMAN

counted for by morphogenetic events at the end of sporulation (16).

DISCUSSION Cyclic AMP phosphodiesterase is one of several proteins of cyclic nucleotide metabolism in Blastocladiella that are found at much higher levels of activity in zoospores than in cells from any other stage of the cell cycle (16, 23, 33; P. Epstein and P. Silvernan, Fed. Proc. 33:1476, 1974). Several observations indicate that these proteins, along with others (22), are synthesized during sporulation as part of a genetic program characteristic of that stage of the Blastocladiella cell cycle. First, zoospores themselves are quiescent with respect to protein synthesis (9, 13, 27, 29). Therefore, zoospore proteins not already present in vegetative cells must be synthesized during sporulation. Specific proteins may also be required for sporulation itself, for example in the formation of a discharge papillum (19) or to provide biosynthetic intermediates in the absence of exogenous nutrients (12). Second, the activities of cyclic AMP phosphodiesterase (see above), cyclic GMP phosphodiesterase (33), guanylate cyclase (23), and alkaline phosphatase (22) all increase during sporulation following a lag period lasting about 60 min. This amount of time is required for protein synthesis to recover from starvation (13), presumably as amino acid pools are restored by protein turnover, and would therefore be expected if enzyme activity were regulated by de novo protein synthesis. Finally, once it has begun, accumulation of cyclic AMP phosphodiesterase activity within single cells requires continuous protein synthesis over an extended interval and most likely reflects synthesis of the phosphodiesterase protein itself. We observe two zoospore components with cyclic AMP phosphodiesterase activity. Each of these has essentially an absolute substrate specificity for.that nucleotide; we could not detect a cyclic AMP phosphodiesterase from any Blastocladiella cell type with significant activity in the hydrolysis of cyclic GMP, in confirmation of previous reports (25, 32), or of other cyclic 3',5'mononucleotides. By this criterion, Blastocladiella enzymes assayed as cyclic AMP phosphodiesterases serve only that function in vivo. The two zoospore components differ in their dimensions, as shown by gel filtration chromatography, and in their stability during storage. The relation between the two components has not, however, been established. They appear not to be the result of rapidly equilibrating species, since each can be prepared essentially free of the other. In view of their comparable substrate

J. BACTERIOL.

specificities, they may be related to each other by posttranslational modification ofa single gene product. To examine the role of hydrolysis in regulating cyclic AMP metabolism in Blastociadiella, we measured the cyclic AMP content of cells over the interval of sporulation when cyclic AMP phosphodiesterase activity increased. Since sporulation is synchronous and involves essentially the entire cell population, any change in total cyclic AMP content could be related at least temporally to phosphodiesterase induction. However, we found no igniicant change in cyclic AMP content over the entire interval. Our interpretation of this surprising result is that the rate of cyclic AMP hydrolysis in sporulating cells is determined not only by the amount of phosphodiesterase, but also by the amount of cyclic AMP substrate available for hydrolysis. The latter could be regulated by cyclic AMP binding proteins, since cyclic AMP bound to such proteins, including one isolated from Bkastocladiella (24), is resistant to phosphodiesterase-catalyzed hydrolysis (5, 20). Significant amounts of tissue cyclic AMP appear to be protein bound, though exact amounts are difficult to quantitate with present methods (11, 30, 31). Beavo et al. (1) have accommodated these facts in the hypothesis that alterations in the amounts of cyclic AMP binding proteins or in their affinity for cyclic AMP could alter the steady-state level of cyclic AMP in a cell without any changes in the rates of cyclic AMP synthesis or hydrolysis. However, if the amount of phosphodiesterase increased along with the amount of bound cyclic AMP so that the absolute rate of hydrolysis remained constant, the result would be a redistribution of cyclic AMP between free and protein-bound pools with no change in the total amount. We have found that the amount of cyclic AMP binding protein increases several fold in sporulating cells in a manner consistent with the operation of this mechanism during sporulation of Biastocladielia (24). Hence, the dynamics of cyclic AMP metabolism in Blastocladiella and presumably the cellular reactions governed by cyclic AMP appear to undergo substantial changes that are not reflected in the total amount of cyclic AMP in the cell. ACKNOMLEDGMENTS We thank Pravina Mehta and Karyl Nat for technical assistance. This work was supported by Public Health Service grants GM-11301 and IT32-GM-07491 from the National Institute of General Medical Sciences and P30-CA-13330 from the National Cancer Institute and by an American Heart Association Established Investigator Award to P.M.S.

VOL. 135, 1978

CYCLIC AMP PHOSPHODIESTERASE IN B. EMERSONII LITERATURE CITED

1. Beavo, J., P. J. Bechtel, and E. G. Krebs. 1974. Activation of protein kinase by physiological concentrations of cyclic AMP. Proc. Natl. Acad. Sci. U.S.A. 71:3680-3583. 2. Boudreau, R. J., and G. I. Drummond. 1975. A modified amay of 3',5'-cyclic AMP phosphodiesterase. Anal. Biochem. 63:388-399. 3. Bourne, H R., G. KL Tomkns, and S. Dion. 1973. Regulation of phosphodiesteraae synthesis requirement for cyclic adenosine monophosphate-dependent protein kinase. Science 181:952-954. 4. Brooker, G., IL Thomas, Jr., and M. KL Appleman.

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Induction of cyclic AMP phosphodiesterase in Blastocladiella emersonii and its relation to cyclic AMP metabolism.

JouRNAL ow BACTRIOLOGY, Sept. 1978, p. 968-975 Vol. 135, No. 3 0021-9193/78/0135-0968$02.00/0 Copyright 0 1978 American Society for Microbiology Pr...
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