Purification and characterization of developmentally regulated AMP deaminase from Dictyostelium discoideum

AMP deaminase, the enzyme that catalyzes the conversion of adenosine monophosphate (AMP) to inosine monophosphate (IMP) and ammonia, was purified from the cellular slime mold, Dictyostelium discoideum in the nutrient-deprived state. The native enzyme had an apparent molecular weight of 199,000 daltons. Its apparent Km was 1.6 mM and its Vmax was 1.0 mumol min-1 mg-1, as measured by the release of IMP From AMP. The enzyme, like other AMP deaminases, was found to be activated by ATP, and inhibited either by GTP or inorganic phosphate. It was also specific for the deamination of AMP. Deaminase activity was increased either when vegetative cells were placed in a nutrient-deprived medium (for up to 6 h) or when vegetative cells were treated with the drug hadacidin. In cells actively growing in complete media, enzyme activity was more non-specific, hydrolyzing adenosine as well as AMP. AMP deaminase in D. discoideum appears to be stage-specific and developmentally regulated, possibly serving to regulate the adenylated nucleotide pool and the interconversion to guanylated nucleotides during early morphodifferentiation.     

Developmental regulation and properties of the cGMP-specific phosphodiesterase in Dictyostelium discoideum

A simple assay has been developed to measure cGMP-specific phosphodiesterase (cGPD) activity in crude soluble extracts of amoebae of Dictyostelium discoideum. When amoebae of different wild-type strains were starved on buffered agar, all strains exhibited an 8- to 12-fold increase in cGMP-specific hydrolyzing activity during development, with the major increase occurring at aggregation. cGMP-specific activity was found in both prestalk and prespore cells. To determine if the elevated cGMP-specific hydrolyzing activity observed during late development was associated with the same enzyme present in vegetative cells, cGMP-specific activities were partially purified from cells at different developmental stages and characterized. Activity in vegetative cells was fractionated by gel filtration into three components with molecular weights of approximately 172,000, 115,000 and 56,000. In contrast, cells starved 4 hr in suspension or 18 hr on agar possessed only the 172,000 or 115,000 Mr forms, respectively. The low-molecular-weight enzyme differed from the two larger forms in kinetic properties and in sensitivity to sulfhydryl reagents. Nevertheless, the three activities probably represent different forms of the same enzyme because mutants defective at the stmF locus lacked appreciable cGMP-specific hydrolyzing activity throughout development. These results indicate that D. discoideum produces a single cGPD which is strongly developmentally regulated. These findings further suggest that intracellular cGMP might be involved in regulating postaggregative as well as preaggregative development.     

Purification and characterization of the NAD-dependent glutamate dehydrogenase in the ectomycorrhizal fungus laccaria bicolor (Maire) orton

The NAD-dependent glutamate dehydrogenase (GDH) (EC 1.4.1.2) from Laccaria bicolor was purified 410-fold to apparent electrophoretic homogeneity with a 40% recovery through a three-step procedure involving ammonium sulfate precipitation, anion-exchange chromatography on DEAE-Trisacryl, and gel filtration. The molecular weight of the native enzyme determined by gel filtration was 470 kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave rise to a single band of 116 kDa, suggesting that the enzyme is composed of four identical subunits. The enzyme was specific for NAD(H). The pH optima were 7.4 and 8.8 for the amination and deamination reactions, respectively. The enzyme was found to be highly unstable, with virtually no activity after 20 days at -75 degrees C, 4 days at 4 degrees C, and 1 h at 50 degrees C. The addition of ammonium sulfate improved greatly the stability of the enzyme and full activity was still observed after several months at -75 degrees C. NAD-GDH activity was stimulated by Ca2+ and Mg2+ but strongly inhibited by Cu2+ and slightly by the nucleotides AMP, ADP, and ATP. The Michaelis constants for NAD, NADH, 2-oxoglutarate, and ammonium were 282 &mgr;M, 89 &mgr;M, 1.35 mM, and 37 mM, respectively. The enzyme had a negative cooperativity for glutamate (Hill number of 0.3), and its Km value increased from 0.24 to 3.6 mM when the glutamate concentration exceeded 1 mM. These affinity constants of the substrates, compared with those of the NADP-GDH of the fungus, suggest that the NAD-GDH is mainly involved in the catabolism of glutamate, while the NADP-GDH is involved in the catalysis of this amino acid. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Isozymes of β-Glucosidase in Dictyostelium discoideum

The activity of beta-glucosidase (EC 3.2.1.21) in extracts of Dictyostelium discoideum was investigated. The specific activity increased early in development, declined during pseudoplasmodium formation, and increased again during sorocarp formation. The beta-glucosidase which was present in growing amoebae and during the first stages of multicellular development was electrophoretically distinct from the enzyme which accumulated during the final stages of morphogenesis. Ribonucleic acid synthesis and protein synthesis during development were required for the accumulation of the later isozyme. Analysis of beta-glucosidase activity in a number of morphological mutants suggests that the enzyme which accumulates late in morphogenesis is developmentally controlled.     

Properties and developmental regulation of the protein phosphatases in Dictyostelium discoideum

The properties and developmental regulation of the protein phosphatases of Dictyostelium discoideum were examined. When crude extracts from vegetative cells were separated on a Mono Q column (FPLC) three protein phosphatase peaks, designated P1, P2 and P3 were found. When aggregation and culmination cells were examined only one protein phosphatase peak was observed. This corresponded to phosphatase P1 of vegetative cells. All three of the vegetative cell phosphatase were inhibited by heparin and mammalian phosphatase inhibitor-2, both of which are specific for type-1 protein phosphatases. Trifluoperazine, which inhibits type-2 protein phosphatases, had little effect on any peaks while levamisole, an alkaline phosphatase inhibitor, stimulated P2, slightly inhibited P3 and had no effect on P1. These results demonstrate the existence of two vegetative phase specific protein phosphatases in D. discoideum and one which occurs during all phases of the life cycle. The protein phosphatases isolated from vegetative cells all appear to be type-1 enzymes.     

Citrate and the regulation of adipose-tissue phosphofructokinase

1. Methods are described for the extraction, partial purification and assay of phosphofructokinase in rat epididymal adipose tissue. 2. The enzyme was inhibited by ATP at concentrations above 20mum and by citrate; the enzyme was activated by ADP, AMP, 3',5'-(cyclic)-AMP, phosphate and sulphate. 3. The enzyme lost activity on incubation at 25 degrees or during chromatography on DEAE-cellulose unless fluoride at a concentration of 4mm was present. 4. The significance of these results in relation to the role of glycolysis and citrate in lipogenesis is discussed.     

AMP-dependence of the cyanide-insensitive pathway in the respiratory chain of Paramecium tetraurelia

The AMP-dependent stimulation of the cyanide-insensitive respiration of Paramecium mitochondria was investigated. The nucleotides exhibiting a stimulatory effect on the cyanide-insensitive oxidation of pyruvate (+ malate) in a medium supplemented with EDTA or carboxyatractyloside were, in decreasing order of efficiency, AMP, GMP, IMP, UMP and TMP. On the other hand, ADP, ATP and cyclic AMP were ineffective. In the presence of carboxyatractyloside, addition of AMP to Paramecium mitochondria incubated with pyruvate (+malate) led to an increase in membrane potential. In the absence of light, the photoactivable derivative of AMP, 3'-[4-[N-(4-azido-2-nitrophenyl)amino]butyryl]-AMP (NAP4-AMP) added to Paramecium mitochondria opposed the stimulatory effect of AMP on the cyanide-insensitive respiration; the Ki for NAP4-AMP was much lower than the Km for AMP, 0.2 microM compared with 120 microM. The ADP-stimulated respiration was not affected. Photoirradiation of Paramecium mitochondria in the presence of NAP4-AMP resulted in irreversible inhibition of the AMP-stimulated cyanide-insensitive respiration. No effect on the ADP-stimulated respiration was observed. A heatlabile cyanide-insensitive ubiquinol oxidase was extracted from Paramecium mitochondria with the detergent NN-dimethyl-N-(3-laurylamidopropyl)amine oxide. The quinol oxidase activity was slightly stimulated by AMP.     

Effects of dithiothreitol on insulin-sensitive phosphodiesterase in rat fat cells

Dithiothreitol activates the low-Km membrane-bound cyclic AMP phosphodiesterase when incubated with the enzyme in a cell-free system. To investigate the mechanism of its activation, we studied the effect of protease inhibitors. Isolated fat cells obtained from Sprague-Dawley rats were incubated in Krebs-Henseleit Hepes buffer, pH 7.4, at 37 degrees C with and without insulin (2 nM, 10 min). A crude microsomal fraction prepared by differential centrifugation was suspended in 0.25 M sucrose containing 10 mM Tes buffer, pH 7.5, with and without 2 mM dithiothreitol and protease inhibitors at 4 degrees C for 48 h. Dithiothreitol stimulated the phosphodiesterase, in a time-dependent manner. As little as 0.02 mM dithiothreitol activated the enzyme, and the maximally effective dose was 2-10 mM. Among the various protease inhibitors tested, antipain, leupeptin, chymostatin and E-64 were the most effective in preventing activation of the enzyme by dithiothreitol. Antipain also inhibited release of the enzyme from the bound fraction. These results suggest that activation of the low-Km phosphodiesterase by dithiothreitol may be provoked by stimulation of an endogenous thiol protease.     

The role of enzyme sequestration in the regulation of the adenylate cyclase of Dictyostelium discoideum

Although the adenylate cyclase of Dictyostelium discoideum cannot be activated by its cAMP agonist in vitro, its in vivo activation can be demonstrated by rapidly breaking and assaying the cells, over 10-fold higher activity being observed for stimulated cells than for basal cells. We report here that when basal cells are broken in the presence of labeled ATP and then rapidly assayed, they display 8-fold more adenylate cyclase activity than cells broken in the presence of unlabeled ATP. This suggests that a significant amount of the enzyme in extracts of basal cells is sequestered within vesicles that can be loaded with substrate at the time of cell lysis, but then rapidly seal. In contrast to the results obtained with basal cells, when cells activated in vivo are broken in the presence of labeled ATP, there is less than 2-fold increase in adenylate cyclase activity. Thus, a much smaller percentage of the observed adenylate cyclase activity of stimulated cells appears to be due to sequestered enzyme than of basal cells. Two models are discussed that account for these observations. One model envisions that roughly equal populations of sequestered and nonsequestered enzyme are produced upon breakage of both basal and activated cells, but that sequestered enzyme in basal extracts becomes uniquely activated in vitro. The other model proposes that the differences in observed activity are due directly to differences in sequestration. According to this latter model, nearly all of the -fold activation previously observed for the D. discoideum adenylate cyclase can be accounted for by a change in sequestration of the enzyme rather than by an intrinsic alteration in the enzyme per se. It therefore suggests a novel mode of regulation whereby an enzyme may be packaged within vesicles and its activity controlled by modulating the permeability of the vesicles to its substrate or effectors.     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Polyphosphate-degrading enzymes in Acinetobacter spp. and activated sludge.

Polyphosphate-degrading enzymes were studied in Acinetobacter spp. and activated sludge. Polyphosphate: AMP phosphotransferase activity in Acinetobacter strain 210A decreased with increasing growth rates. The activity of this enzyme in cell extracts of Acinetobacter strain 210A was maximal at a pH of 8.5 and a temperature of 40 degrees C and was stimulated by (NH4)2SO4. The Km for AMP was 0.6 mM, and the Vmax was 60 nmol/min per mg of protein. Cell extracts of this strain also contained polyphosphatase, which was able to degrade native polyphosphate and synthetic magnesium polyphosphate and was strongly stimulated by 300 to 400 mM NH4Cl. A positive correlation was found between polyphosphate:AMP phosphotransferase activity, adenylate kinase activity, and phosphorus accumulation in six Acinetobacter strains. Significant activities of polyphosphate kinase were detected only in strain P, which contained no polyphosphate:AMP phosphotransferase. In samples of activated sludge from different plants, the activity of adenylate kinase correlated well with the ability of the sludge to remove phosphate biologically from wastewater.     

Subcellular distribution and movement of 5''-nucleotidase in rat cells.

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.     

Studies on the nature of the regulation by purine nucleotides of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase from Ehrlich ascites-tumour cells

1. The progress curves of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase activity plotted against 5-phosphoribosyl pyrophosphate concentration were hyperbolic in nature. The inhibition of the former enzyme by AMP and GMP and of the latter enzyme by IMP and GMP showed completely competitive characteristics. 2. The effect of temperature on the reaction of adenine phosphoribosyltransferase and of hypoxanthine phosphoribosyltransferase was examined. The energy of activation of the former enzyme decreased at temperatures greater than 27 degrees and that of the latter enzyme at temperatures greater than 23 degrees . For each enzyme, the change in the heat of formation of the 5-phosphoribosyl pyrophosphate-enzyme complex at the critical temperature was approximately equal to the change in the energy of activation but was in the opposite direction. The inhibitor constants with both enzymes in the presence of nucleotides varied in different ways with temperature from the Michaelis constants for 5-phosphoribosyl pyrophosphate indicating that different functional groups were involved in binding substrates and inhibitors. 3. ATP was found to stimulate adenine-phosphoribosyltransferase activity at concentrations less than about 250mum and to inhibit the enzyme at concentrations greater than 250mum. The stimulation was unaffected by 5-phosphoribosyl pyrophosphate concentration but the inhibitory effect could be overcome by increasing concentrations of this compound. At low concentrations ATP reversed the inhibition of adenine phosphoribosyltransferase by AMP and GMP to an extent dependent on their concentration. 4. The properties of adenine phosphoribosyltransferase changed markedly on purification. Crude extracts of ascites-tumour cells had Michaelis constants for 5-phosphoribosyl pyrophosphate and adenine 75 and six times as high respectively as those obtained with purified enzyme. ATP had no stimulatory effect on activity of the purified enzyme or on that of crude extracts heated 15min. or longer at 55 degrees . 5. It is suggested that at low concentrations ATP is bound to an ;activator' site which is separate from the substrate binding site of adenine phosphorytransferase and that at high concentrations ATP competes with 5-phosphoribosyl pyrophosphate at the active site of the enzyme.     

A study of the role of glutamate dehydrogenase in the nitrogen metabolism of Arabidopsis thaliana

Glutamate-dehydrogenase (GDH, EC 1.4.1.2) activity and isoenzyme patterns were investigated in Arabidopsis thaliana plantlets, and parallel studies were carried out on glutamine synthetase (GS, EC 6.3.1.2). Both NADH-GDH and NAD-GDH activities increased during plant development whereas GS activity declined. Leaves deprived of light showed a considerable enhancement of NADH-GDH activity. In roots, both GDH activities were induced by ammonia whereas in leaves nitrogen assimilation was less important. It was demonstrated that the increase in GDH activity was the result of de-novo protein synthesis. High nitrogen levels were first assimilated by NADH-GDH, while GS was actively involved in nitrogen metabolism only when the enzyme was stimulated by a supply of energy, generated by NAD-GDH or by feeding sucrose. When methionine sulfoximine, an inhibitor of GS, was added to the feeding solution, NADH-GDH activity remained unaffected in leaves whereas NAD-GDH was induced. In roots, however, there was a marked activation of GDH and no inactivation of GS. It was concluded that NADH-GDH was involved in the detoxification of high nitrogen levels while NAD-GDH was mainly responsible for the supply of energy to the cell during active assimilation. Glutamine synthetase, on the other hand was involved in the assimilation of physiological amounts of nitrogen. A study of the isoenzyme pattern of GDH indicated that a good correlation existed between the relative activity of the isoenzymes and the ratio of aminating to deaminating enzyme activities. The NADH-GDH activity corresponded to the more anodal isoenzymes while the NAD-GDH activity corresponded to the cathodal ones. The results indicate that the two genes involved in the formation of GDH control the expression of enzymes with different metabolic functions.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - MSO methionine sulfoximine     

Guanidine hydrochloride-induced shedding of a Dictyostelium discoideum plasma membrane fraction enriched in the cyclic adenosine 3',5'-monophosphate receptor

The cell surface cyclic AMP receptor of Dictyostelium discoideum is under study in a number of laboratories with respect to both its role in development of the organism and the physiology of excitation-response coupling. We report here that when starved amoebae are exposed to the chaotrope guanidine hydrochloride at 1.8 M, they shed a particulate cyclic AMP binding activity into the medium. This activity is due to membrane vesicles which originate from the cell surface. The vesicles are enriched up to 150-fold in cyclic AMP binding activity and up to 14-fold in phospholipid content when compared to the starting amoebae. The cyclic AMP binding activity of the membrane vesicles is identical to that of the cell surface receptor with respect to the following properties; (i) it is lacking in preparations from unstarved, vegetative amoebae; (ii) it is not inhibited by cyclic GMP and is stimulated by calcium ions; (iii) it has very rapid rates of association and dissociation of bound cyclic AMP; (iv) it has two classes of binding sites with dissociation constants similar to those of the surface receptors of whole amoebae. The binding activity of the isolated membranes is stable for several days at 4 degrees C and the lower affinity binding sites are stable up to several months when stored at -80 degrees C. Due to enrichment and stability of the receptor in this preparation, it should be highly suitable for many types of studies. The usefulness is enhanced by the fact that the preparation does not contain detectable cyclic AMP phosphodiesterase activity.     

De novo purine synthesis in vegetative cells and myxospores of Myxococcus xanthus

This study was designed to determine whether vegetative cells and myxospores of Myxococcus xanthus were capable of classical de novo purine biosynthesis. To answer this question, vegetative and myxospore extracts of M. xanthus FBa were tested for their ability to synthesize the second de novo intermediate, 5'-phosphoribosylglycinamide, from beginning precursors either by way of phosphoribosyl-pyrophosphate amido transferase (EC 2.4.2.14) or ribose-5-phosphate amino transferase. Both the amido and amino transferase routes occurred in both types of extracts, and both enzymes appear to be present at about the same level (per milligram of protein) in vegetative cells, myxospores, and in a bacterial prototype, Salmonella typhimurium. The dose response of the vegetative and myxospore forms of both enzymes towards adenosine 5'-monophosphate (AMP) and guanosine 5'-monophosphate (GMP) suggests that the allosteric structure of both enzymes is changed little by sporulation. Both enzymes were inhibited to varying degrees by a variety of purine nucleotides besides AMP, GMP, and 3':5' cyclic AMP.     

Evidence for a membrane-bound pyrophosphatase in Dictyostelium discoideum

Plasma membrane enriched fractions of Dictyostelium discoideum contain a Des-insensitive ATPase activity that can be fractionated by DEAE-Sephacel into a major vanadate-sensitive activity and a minor vanadate-insensitive activity. The vanadate-insensitive activity hydrolyzed pyrophosphate considerably more rapidly than ATP or any other substrate tested, and the enzyme was therefore designated a pyrophosphatase. The enzyme had no activity on AMP or p-nitrophenyl phosphate. The pyrophosphatase activity was maximal at alkaline pH values and stimulated by Mg2+ but not by Ca2+, properties of the enzyme that are very similar to those of the previously characterized pyrophosphatases of the plant tonoplast membrane. The pyrophosphatase activity of total membrane extracts changed very little during Dictyostelium differentiation.     

Structural requirements for the binding of AMP to the allosteric site of NAD-specific isocitrate dehydrogenase from bakers' yeast

The specificity of yeast NAD-specific isocitrate dehydrogenase for the structures of the allosteric effector 5'-AMP was examined with analogues modified in the purine ring, pentosyl group, and 5'-phosphate group. An unsubstituted 6-amino group was essential for activation as was the phosphoryl group at the 5'-position. Activity was retained when an oxygen function of the 5'-phosphoryl was replaced by sulfur (Murry & Atkinson, 1968) or by nitrogen (phosphoramidates). 2-NH2-AMP, 2-azido-AMP, and 8-NH2-AMP were active; 8-azido-AMP and 8-Br-AMP were inactive. The configuration or nature of substituents about carbons 2' and 3' of the pentosyl portion of AMP was not critical for allosteric activation since AMP analogues containing, e.g., 2',3'-dideoxyribose or the bulky 2',3'-O-(2,4,6-trinitrocyclo-hexadienylidene) substituent (TNP-AMP) were active. TNP-AMP was bound to the enzyme with fluorescence enhancement and had an S0.5 for activation similar to the S0.5 for AMP. Positive effector activity was decreased when the pentosyl moiety of 5'-AMP was replaced by the six-membered nitrogen-containing morpholine group, indicating that the pentosyl group may be critical as a spacer for the proper geometry of binding to enzyme at the 6-amino and 5'-phosphoryl groups of 5'-AMP. A comparison of molecular models of 5'-AMP with 8,5'-cycloAMP suggests that the species of 5'-AMP required for binding to the enzyme contains the purine and ribose moieties in an anti conformation and positioning of the 5'-phosphate trans with respect to carbon 4'.(ABSTRACT TRUNCATED AT 250 WORDS)     

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

Extracts of vegetative cells 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 examine the relation between enzyme synthesis and cyclic nucleotide metabolicm, 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.