Purification and properties of a cyclic-AMP phosphodiesterase that is active in only one cell type during the multicellular development of Dictyostelium discoideum

Cyclic-AMP phosphodiesterase (PDE) accumulates during the aggregation stage of Dictyostelium where it functions in maintaining extracellular levels of cyclic AMP (cAMP). The activity decreases during the subsequent multicellular slug stage and then accumulates again during sorocarp construction, but the enzyme is active only in the developing stalk. Because of the possible significance of this localized activity in only one of the two cell types, we have purified the enzyme from the multicellular stage in order to understand its mode of regulation in vivo. We find that the enzyme which is localized in the prestalk cells is similar in many respects to the extracellular PDE which is active at the aggregation stage. The enzyme from both stages is inhibited by a low molecular weight protein. The mechanism of this inhibition is through a shift in the apparent Km for cAMP from micromolar to millimolar levels. The inhibited form of the enzyme can be activated by preincubation with MgSO4 and dithiothreitol (DTT). This activation treatment releases the inhibitor from the enzyme, thus restoring the low Km form, changes the molecular weight of the culmination stage enzyme from 95 000-100 000 to 68 000 by releasing the Mr 35 000-40 000 inhibitor protein, and causes irreversible loss of inhibitor activity. Although the inhibitor could be obtained in high yield from the aggregation stage by simply heating the extracellular fluid, it could not be detected from culmination stage extracts when prepared by this method. However, inclusion of calcium in the extraction buffer resulted in release of inhibitor from both heated and nonheated samples. The results indicate that the stalk cell specific PDE is regulated similarly to the aggregation stage PDE and opens the possibility of differential regulation of PDE in the two cell types.     

Separation and investigation of the regulatory properties of two forms of cyclic nucleotide phosphodiesterase from rabbit heart--sensitive and insensitive to Ca-dependent regulator protein]

Two forms of cyclic nucleotide phosphodiesterase (ES 3.1.4.17)--PDE-I and PDE-II--sensitive and resistant to Ca-dependent protein regulator, were isolated from the soluble fraction of rabbit heart by chromatography on DEAE-cellulose. Both forms of enzyme are inhibited by 30--50% by Ca2+ (10(-4) M). Addition of Ca-dependent protein regulator activates PDE-I and eliminates Ca2+-induced inhibition of PDE-II. In heart extract Ca2+ increases the phosphodiesterase activity 1.5-fold. The amount of PDE-I makes up to about 10% of total phosphodiesterase activity of the heart; that of PDE-II is about 90%. In the presence of Ca-dependent protein regulator the rate of 3', 5'-AMP hydrolysis by PDE-I is increased 5--15-fold, while that of 3', 5'-GMP hydrolysis only 2.5-fold. Both PDE-I and PDE-II have close Km values for substrates--(3.5--4.0).10(-6) M for 3', 5'-AMP and 14.10(-6) M for 3', 5'-GMP. Inhibition by Ca2+ and effect of Ca-dependent protein regulator manifest themselves in changes in V for cyclic nucleotide hydrolysis and do not alter the Km value for the enzyme.     

The isoenzyme selectivity of AH 21-132 as an inhibitor of cyclic nucleotide phosphodiesterase activity.

The smooth muscle relaxant, AH 21-132, was tested for its inhibitory effect on the cyclic nucleotide phosphodiesterase (PDE) activities fractionated from guinea-pig cardiac ventricle and bovine trachealis muscle. Both tissues yielded significant PDE-I and PDE-II activities. The cardiac ventricle also contained a significant amount of PDE-III whilst the trachealis contained PDE-IV. AH 21-132 inhibited PDE-III and PDE-IV selectively (Ki values 0.30-0.55 microM) compared with PDE-I and PDE-II (Ki values 20-140 microM).     

Localization of glycogen synthetase during differentiation of presumptive cell types in Dictyostelium discoideum.

Ultramicrochemical techniques were utilized to assay glycogen synthetase (EC 2.4.1.11) activity in cell samples of Dictyostelium discoideum as small as 0.01 mug (dry weight) in reaction volumes of 0.1 mul. The activity was assayed by an amplification procedure employing the enzymatic cycling of pyridine nucleotides. These techniques were used to determine the extent of localization of glycogen synthetase in the two cell types during differentiation of D. discoideum. Localization studies in developing spore cells revealed decreasing enzyme activity to the culmination stage. During this phase of development, the enzyme required the presence of soluble glycogen for activity. From culmination to sorocarp stage, enzyme activity increased and was independent of the soluble glycogen. In developing stalk cells, synthetase showed a decreasing gradient of activity. In sorocarps, the cells in the stalk apex showed synthetase activity similar to that of the spores. The cells at the bottom of the stalk had no detectable activity.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

A genetic melanotic neoplasm of Drosophila melanogaster

The construction of mature fruiting bodies occurs during the culmination stage of development of Dictyostelium discoideum. These contain at least two different cell types, spores and stalks, which originate from an initially homogenous population of vegetative amoebas. As an attempt to identify proteins whose synthesis is regulated in each cell type during differentiation, we have analyzed the two-dimensional profiles of proteins synthesized by spore and stalk cells during the culmination stage. We have identified 5 major polypeptides which are specifically synthesized by spore cells during culmination and 9 which are only made by stalk cells. Furthermore, synthesis of about 20 polypeptides appears to be enriched either in the spore or in the stalk cells. We also show that synthesis of actin, a major protein synthesized during Dictyostelium development, is specifically inhibited in the spore cells during culmination. Synthesis of most of the cell type-specific proteins initiates at 19–20 hr, during culmination. Moreover, the proteins whose synthesis is induced after formation of tight aggregates, the time when the major change in gene expression occurs, are not specifically incorporated into spores or stalk cells, and appear to be synthesized by both cell types. We conclude that a new class of genes is expressed during the culmination stage in Dictyostelium, giving rise to specific patterns of protein synthesis in spore and stalk cells.     

Localization of cyclic nucleotide phosphodiesterase in the multicellular stages of Dictyostelium discoideum

Cyclic 3',5'-adenosine monophosphate (cAMP) is secreted as the chemotactic signal by aggregating amoebae of the cellular slime mold Dictyostelium discoideum. We have used ultramicrotechniques in the biochemical analysis of cyclic nucleotide phosphodiesterase (PD) distribution in individual aggregates at various stages of development. With handmade constriction pipettes in microliter volumes, sections of lyophilized individuals weighing 20-100 ng could be assayed in a reaction coupled to 5'-nucleotidase. Phosphodiesterase activity was measured at pH 7.5 with 12 microM cAMP, cAMP-PD activity in aggregates ranged from 20-40 mmol/h/kg. In the pseudoplasmodium it had dropped to 5-10 mmol/h/kg and a difference in activity between the anterior prestalk cells and posterior prespore cells began to appear. The utmost posterior sections showed elevated phosphodiesterase from this stage onward. During culmination, activity rose to 40-60 mmol/h/kg associated with the developing stalk, while it declined in the spore mass. The papilla remained constant at 5-10 mmol/h/kg. The pattern of localization in the stalk was the same when cGMP was used as substrate. Extracellular phosphodiesterase inhibitor produced at the aggregation stage was found to reduce the localized activity in the culmination stage by 50-80%, with the most marked inhibition occurring in the center of the papilla. We found no evidence of endogenous heat-stable phosphodiesterase inhibitor within the culminating sorocarp.     

Extracellular cGMP phosphodiesterase related to the rod outer segment phosphodiesterase isolated from bovine and monkey retinas

A phosphodiesterase (PDE) has been characterized in the interphotoreceptor matrix (IPM) of light-adapted fresh bovine retinas. It is obtained through a gentle rinsing of the retinal surface under conditions where the light-activated rod outer segment (ROS) enzyme remains attached. The enzyme has an apparent native molecular weight of 350 000 by gel filtration and appears as a doublet at Mr 47 000 and 45 000 on sodium dodecyl sulfate-polyacrylamide gels. It has an apparent Km value for cGMP of 33 microM and an apparent Km value for cAMP of 2200 microM. It is activated 3-6-fold by protamine and over 40-fold by trypsin. Protamine has no effect on the Km for cGMP while trypsin decreases the Km for cGMP by a factor of 2. The enzyme occurs in at least two forms as evidenced by two distinct peaks of activity after gel electrophoresis under nondenaturing conditions. A heat-stable inhibitor is tightly bound to the enzyme. The inhibitor obtained from the IPM PDE inhibits 98% of the activity of the trypsin-activated ROS PDE: conversely, the inhibitor obtained by boiling the ROS PDE completely inhibits the trypsin-activated IPM enzyme. A high-affinity monoclonal antibody to the active site of the ROS PDE, ROS 1 [Hurwitz, R., Bunt-Milan, A.H., & Beavo, J. (1984) J. Biol. Chem. 259, 8612-8618], quantitatively absorbs the IPM PDE. These observations indicate a clear relationship between these two PDEs even though their location, sizes, and specific functions in the retina appear to be distinct.     

Direct photolabeling of the cGMP-stimulated cyclic nucleotide phosphodiesterase

cGMP-stimulated phosphodiesterase (PDE) has been directly photolabeled with [32P]cGMP using UV light. Sequence analysis of peptide fragments obtained from partial proteolysis or cyanogen bromide cleavage indicate that two different domains are labeled. One site, on a Mr = 36,000 chymotryptic fragment located near the COOH terminus, has characteristics consistent with it being close to or part of the catalytic site of the enzyme. This peptide contains a region of sequence that is highly conserved in all mammalian cyclic nucleotide PDEs and has been postulated to contain the catalytic domain of the enzyme. The other site, on a Mr = 28,000 cyanogen bromide cleavage fragment located near the middle of the molecule, probably makes up part of the allosteric site of the molecule. Labeling of the enzyme is concentration dependent and Scatchard analysis of labeling yields a biphasic plot with apparent half labeling concentrations of about 1 and 30 microM consistent with two types of sites being labeled. Limited proteolysis of the PDE by chymotrypsin yields five prominent fragments that separate by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at Mr = 60,000, 57,000, 36,000, 21,000, and 17,000. Both the Mr = 60,000 and 57,000 apparently have blocked NH2 termini suggesting that the Mr = 57,000 fragment is a subfragment of the Mr = 60,000 fragment. Primary sequence analysis indicates that both the Mr = 21,000 and 17,000 fragments are subfragments of the Mr = 36,000 fragment. Autoradiographs of photolabeled then partially proteolyzed enzyme show labeled bands at Mr = 60,000, 57,000, and 36,000. Addition of 5 microM cAMP prior to photolabeling eliminates photolabeling of the Mr = 36,000 fragment but not the Mr = 60,000 or 57,000 fragments. The labeled site not blocked by cAMP is also contained in a Mr = 28,000 cyanogen bromide fragment of the enzyme that does not overlap with the Mr = 36,000 proteolytic fragment. Limited chymotryptic proteolysis also increases basal activity and eliminates cGMP stimulation of cAMP hydrolysis. The chymotryptic fragments can be separated by either ion exchange high performance liquid chromatography (HPLC) or solid-phase monoclonal antibody treatment. A solid-phase monoclonal antibody against the cGMP-stimulated PDE removes the Mr = 60,000 and 57,000 labeled fragments and any intact, unproteolyzed protein but does not remove the Mr = 36,000 fragment or the majority of activity. Ion exchange HPLC separates the fragments into three peaks (I, II, and III). Peaks I and II contain activity of approximately 40 and 100 units/mg, respectively. Peak II is the undigested or slightly nicked native enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cell specific activity of trehalose-6-phosphate synthetase during differentiation of Dictyostelium discoideum.

Trehalose-6-P synthetase activity was low at the beginning of the life cycle of Dictyostelium discoideum, reached maximum activity at 20 h, and decreased at late sorocarp. Enzyme activity in developing spore cells increased 10-fold during differentiation from myxamoebae (0 h) to the culmination stage (20 h) and decreased slightly at sorocarp (24 h). Activity was similar in spore cells at the apex of the stalk. The activities in the stalk cells were dependent upon their position in the developing stalk. There was a decreasing gradient of activity from the apex to the base of the stalk.     

The isolation and characterization of cyclic nucleotide phosphodiesterases from Morris hepatoma 5123tc(h) and rat liver

Four main phosphodiesterase (PDE) forms were resolved and partially purified from rat liver and Morris hepatoma 5123tc(h). The activities of the high Km cyclic nucleotide PDE (form II) in hepatoma were markedly reduced compared to liver, while the activities of the low Km cAMP PDE (form III) and low Km cyclic nucleotide PDE (form IV) in hepatoma were markedly higher than those of liver. The partially purified low Km cAMP PDE's (forms III and IV) from liver showed non-linear Lineweaver-Burk plots, whereas the same enzyme forms in hepatoma displayed linear kinetics. Activation of low Km cGMP PDE activity by calmodulin was found with form I in liver whereas in hepatoma form II was responsive to calmodulin.     

Culmination in Dictyostelium is regulated by the cAMP-dependent protein kinase.

We placed a specific inhibitor of cyclic AMP-dependent protein kinase (PKA) under the control of a prestalk-specific promoter. Cells containing this construct form normally patterned slugs, but under environmental conditions that normally trigger immediate culmination, the slugs undergo prolonged migration. Slugs that eventually enter culmination do so normally but arrest as elongated, hairlike structures that contain neither stalk nor spore cells. Mutant cells do not migrate to the stalk entrance when codeveloped with wild-type cells and show greatly reduced inducibility by DIF, the stalk cell morphogen. These results suggest that the activity of PKA is necessary for the altered pattern of movement of prestalk cells at culmination and their differentiation into stalk cells. We propose a model whereby a protein repressor, under the control of PKA, inhibits precocious induction of stalk cell differentiation by DIF and so regulates the choice between slug migration and culmination.     

Expression of adenylate cyclase and phosphodiesterase in development of temperature-sensitive mutants with impaired metabolism of cAMP in drosophila melanogaster

The mode of the developmental expression of adenylate cyclase (AC) and phosphodiesterase (PDE) in D melanogaster indicates that PDE plays the major role in the maintenance of a certain level of cAMP in postembryonic development, while both enzymes function in concert in imago. The ts-mutants ts155 and ts622, characterized upon their isolation as having an increased cAMP content and normal PDE activity, manifest high levels of AC activity from the third day of imago life. The levels of PDE activity characteristic for adult mutants with altered enzyme activity (low in ts66 and ts980, high in ts398) are manifested in ts980 from larval instar II, and from the larval instar III in ts398 and ts66. Data on the dependence of PDE activity in adults upon temperature of incubation, being in agreement with the expectations for a ts-mutation in a gene coding for a form of PDE in case of ts66, suggest that ts398 affects not the enzyme-coding gene but rather one for an activator protein. The fact that in ts398 (the polyphasic ts-lethal mapping to 1-38.9) 1) AC activity is somewhat higher than normal at 22°C and is readily activated at 29°C, 2) activity of PDE-I assayed in heat-pretreated homogenates is higher than normal, 3) that boiled extracts of ts398 are potent activators of the wild type and of its own PDE-I indicates that it is a mutation affecting calmodulin, which is known to be stable at boiling and capable of activating both AC and PDE-I. Data on Ca²⁺ and EGTA effects suggest that the mutation presumably increases Ca²⁺-binding activity of calmodulin, ts980 and ts622, in which ts-lethality could be produced only by certain doses of haloperidol and triftazine, appear to be lethal in compounds with ts398, thus indicating that these mutations could affect the same calmodulin-controlling gene.     

Localization of glycogen phosphorylase in specific cell types during differentiation of Dictyostelium discoideum

The localization of glycogen phosphorylase was studied during the differentiation of prespore and prestalk cells in Dictyostelium discoideum. Ultramicrotechniques were utilized to assay the enzyme activity in cell samples as small as 0.02 μg dry wt in reaction volumes of 0.1 μl. The activity was assayed using an amplification procedure employing the enzymatic cycling of pyridine nucleotides. Glycogen phosphorylase from individual organisms was assayed during the developmental period. Early in development, activity was low but gradually increased to a maximum value at culmination. From culmination to sorocarp, enzyme activity decreased rapidly. Cell-specific assays of spores showed that phosphorylase activity increased slightly to culmination, and then decreased. Prestalk cells showed the greatest activity in the area of stalk sheath construction and elongation. Stalk cells showed a decreasing gradient of enzyme activity from the tip of the stalk to the base. Enzyme activity in the spores may be sufficient to provide glucose units for trehalose synthesis and spore coat production. The prestalk enzyme may degrade glycogen to provide glycosyl units for production of the stalk sheath and trehalose. Possible models of cell-specific biochemical events in Dictyostelium discoideum are discussed.     

Some features of cyclic adenosine monophosphate metabolism in mouse liver and hepatoma 22]

The levels of cyclic adenosine monophosphate (cAMP) and two forms of cAMP phosphodiesterase with low (PDE1) and high (PDE2) affinity for the substrate were determined in homogenates from mouse liver and transplanted hepatoma 22. The level of cAMP in the tumour is 3 times lower than that in liver. By te kinetic parameters (Vmax, Km, pH optimum) adenylate cyclase from tumour does not show any significant differences as compared to the liver enzyme; the enzyme from hepatoma is, however, more sensitive to activation by F- ions. The activities of adenylate cyclase in liver and tumour cells are the same. Phosphodiesterases of cAMP from tumour and liver cells are similar in their Km values (3,3-10(-4) M for PDE1 and 2-10(-6) M for PDE2); however, the maximal and real rates of cAMP hydrolysis in hepatoma are much higher than in liver. The fact that both cAMP phosphodiesterase activities have similar dependence on Mg2+ and Ca2+ concentrations, suggests that PDE1 is a latent form of PDE2. In tumour cells the equilibrium between these two forms is probably shifted towards the enzyme with high affinity for the substrate. The results suggest that a decreased cAMP level in hepatoma cells (as compared to the liver) is due to the activation of PDE2.     

The timing of cell-type-specific differentiation in Dictyostelium discoideum

We have used two-dimensional gel electrophoresis to identify over 30 proteins which are specific to one or other of the two cell types of Dictyostelium discoideum, either at the slug stage or in mature fruiting bodies. Our results support the idea that there is a continuous developmental program that begins in prespore cells at the hemispherical mound stage (10-12 hr) and results in spore differentiation (24 hr). Prestalk differentiation, on the other hand, appeared largely unrelated to stalk differentiation, which was first detectable at the onset of culmination (18 hr). We have also used this approach to study the differentiation of stalk-only mutants and have found that the cells can switch from spore to stalk differentiation as late as 2 hr before the end of the wild-type developmental program.     

Glycogen degradation during migration of presumptive cell types in Dictyostelium discoideum.

During the time course of differentiation in Dictyostelium discoideum, glycogen was found to accumulate from the amoebae stage to the culmination stage of development. Upon sorocarp formation (23 h), glycogen was rapidly degraded. Ultramicrotechniques, utilizing amplification of glycogen by enzymatic cycling, were used to follow glycogen metabolism in pre-stalk and prespore cells during the differentiation cycle. Both cell types accumulated glycogen at nearly the same rate. By the pseudoplasmodium stage of development glycogen had accumulated to 50% of its maximum value, and no differences were found between pre-stalk and pre-spore cells. Glycogen was degraded as pre-stalk cells migrated into the position for stalk construction. At the culmination stage of development stalk cells near the base were devoid of glycogen while pre-stalk cells near the apex of the stalk showed no loss of glycogen. The complete loss of glycogen from stalk cells occurred over a distance occupied by approximately 100 cells, and over a time period of approx. 1 h. Pre-spore cells at the culmination stage showed no loss of glycogen even though separated from stalk cells by only a thin cellulose sheath. The degradation of prespore cell glycogen did not commence until stalk construction was completed and the pre-spore mass had reached the apex of the stalk. Pre-spore cells at the culmination stage contained high levels of glycogen while only 2 h later, total degradation had occurred.     

A prespore gene, Dd31, expressed during culmination of Dictyostelium discoideum

During culmination of Dictyostelium fruiting bodies, prespore and prestalk cells undergo terminal differentiation to form spores and a cellular stalk. A genomic fragment was isolated by random cloning that hybridizes to a 1.4-kb mRNA present during culmination. Cell type separations at culmination showed that the mRNA is present in prespore cells and spores, but not in prestalk or stalk cells. After genomic mapping, an additional 3 kb of DNA surrounding the original 1-kb fragment was cloned. The gene was sequenced and named Dd31 after the size of the predicted protein product in kilodaltons. Accumulation of Dd31 mRNA occurs immediately prior to sporulation. Addition of 20 mM 8-Br-cAMP to cells dissociated from Mexican hat stage culminants induced sporulation and the accumulation of Dd31 mRNA, while 20 mM cAMP did not. Dd31 mRNA does not accumulate in the homeotic mutant stalky in which prespore cells are converted to stalk cells rather than spores. Characterization of Dd31 extends the known temporal dependent sequence of molecular differentiations to sporulation.     

Activation of the prespore and spore cell pathway of Dictyostelium differentiation by cAMP-dependent protein kinase and evidence for its upstream regulation by ammonia.

Expression of a dominant inhibitor of the Dictyostelium cAMP-dependent protein kinase in prespore cells blocks their differentiation into spore cells. The resultant structures comprise a normal stalk supporting a bolus of cells that fail to express a sporulation-specific gene and that show greatly reduced levels of expression of several prespore-specific genes. The latter result suggests that in addition to activating spore formation, the cAMP-dependent protein kinase may play a role in initial prespore cell differentiation. Development of the strain expressing the dominant inhibitor is hypersensitive to the inhibitory effects of ammonia, the molecule that is believed to repress entry into culmination during normal development. This result supports a model whereby a decrease in ambient ammonia concentration at culmination acts to elevate intracellular cAMP and hence induce terminal differentiation.     

Localization of adenylate cyclase during development of Dictyostelium discoideum

Cyclic AMP is known to function as the chemotactic signal during aggregation of single-celled amoebae of the cellular slime mold Dictyostelium discoideum. Evidence from several laboratories has accumulated suggesting that cAMP also acts as a regulatory molecule during Dictyostelium multicellular differentiation. We have used ultramicrotechniques and a sensitive radioimmunoassay in the localization of adenylate cyclase, the cAMP synthetic enzyme, during the development of Dictyostelium. We demonstrate that adenylate cyclase activity is localized in the prespore cells of the culminating individual with no activity detectable in the prestalk region. We show that this lack of activity in the stalk may be due to a masking by an endogenous inhibitor of the enzyme. Within the spore mass we found an increasing gradient of enzyme activity toward the base. These data, along with that from the localization of cyclic nucleotide phosphodiesterase, indicate that an enzymatic potential exists for the creation of cAMP gradients during development in the organism. Such a gradient may provide positional information necessary to direct the terminal differentiation of spore and stalk cells.