Effect of thymalin on the cyclic nucleotide system in the mouse spleen

The effect of the thymus preparation thymalin (1000 micrograms/mouse, i.p.) on the system of cyclic nucleotides in the spleen has been studied. The preparation produced two-phasic changes in the content of cAMP and cGMP, as well as in the activity of cAMP phosphodiesterase. The changes in the spleen cAMP content are consequent on those in the activity of phosphodiesterase.     

Regulation of cyclic nucleotide phosphodiesterases in cultured hepatoma cells by dexamethasone and N6,O2'-dibutyryl adenosine 3':k'-monophosphate.

DEAE-Bio-Gel chromatography of 100,000 X g supernatant from cultured HTC hepatoma cells separated cyclic nucleotide phosphodiesterase into three forms, numbered E I, E II, and E III in order of elution from the column, E I had a low Km for cyclic guanosine 3':5'-monophosphate (cGMP) and a high Km for cyclic adenosine 3':5'-monophosphate (cAMP), E II exhibited anomalous kinetics. At low substrate concentrations (0.5 muM) cGMP was hydrolyzed more rapidly than cAMP and hydrolysis of 0.5 muM cAMP was stimulated by 1 muM cGMP. E III had a low Km for cAMP. Incubation of cells with 1 muM dexamethasone for 72 h decreased the activity of E I and E II. In cells incubated with N6,O2'-dibutyryl cAMP plus 3-isobutyl-1-methylxanthine for 14 h the activity of E III was increased approximately 100%. Similar activities of calcium-dependent, heat stable phosphodiesterase activator were recovered from supernatants from all cells. These studies have established the presence, in a homogeneous population of hepatoma cells, of at least three forms of cyclic nucleotide phosphodiesterase, the activities of which can be independently regulated.     

Differential susceptibility to biological detergents of the particulate cGMP-stimulated phosphodiesterase from rat heart: preservation of the allosteric properties of the solubilized enzyme

The effects of various biological detergents on the particulate cGMP-stimulated cAMP phosphodiesterase activity from rat heart were investigated. When added to particulate fractions, anionic and non-ionic detergents diversely increased both cAMP and cGMP phosphodiesterase activities and slightly decreased the stimulatory effect of cGMP on cAMP hydrolysis whereas cationic detergents were rather inhibitory and drastically lowered the stimulatory effect of cGMP. Among the most efficient detergents, only sodium cholate was able to solubilize phosphodiesterase activity and preserve the stimulatory effect of cGMP on cAMP hydrolysis. Furthermore, the addition of glycerol significantly improved the conservation of the allosteric properties of the enzyme. Kinetic properties of the cholate-solubilized phosphodiesterase were quite identical to those of the membrane-bound enzyme.     

Differences and similarities between guanosine 3',5'-cyclic monophosphate phosphodiesterase and adenosine 3',5'-cyclic monophosphate phosphodiesterase activities in neuroblastoma cells in culture.

There are phosphodiesterase activities in both particulate and supernatant fractions which hydrolyze guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) with an apparent Km of 2-8 muM and with an apparent Km of 44-222 muM. 4-(3-Butoxy-4-methoxybenzyl-2-imidazolidinone (RO20-1724) did not inhibit cGMP phosphodiesterase activity in homogenates of mouse neuroblastoma cells, but markedly inhibited cAMP phosphodiesterase activity. Papaverine and theophylline inhibited both cGMP and cAMP phosphodiesterase activities to about the same extent. The former was more potent than the latter. The specific activity of cGMP phosphodiesterase as a function of protein concentrations first increased and then decreased. The specific activity of cAMP phosphodiesterase decreased under a similar experimental condition.     

Dissimilar cyclic nucleotide phosphodiesterase activities in subcellular fractions from normal and SV40-transformed WI-38 fibroblasts.

Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.     

Characterization of soluble uterine cyclic nucleotide phosphodiesterase.

Soluble cyclic nucleotide phosphodiesterase of rat uterus displays distinct structural and regulatory properties. Like phosphodiesterases from many mammalian sources the soluble uterine enzyme system exhibits nonlinear Lineweaver--Burk kinetics with cyclic adenosine 3':5'-monophosphate (cAMP) as substrate (apparent Kms congruent to 3 and 20 micron) and linear kinetics with cyclic guanosine 3':5'-monophosphate (cGMP) as substrate (apparent Km congruent to 3 micron). Unlike most other mammalian phosphodiesterases, however, numerous separation procedures reveal only a single form of uterine phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP. A single form of the enzyme is observed upon sucrose gradient centrifugation (7.9 S), agarose gel filtration, and DEAE-cellulose chromatography at either pH 8.0 OR 6.0. Heat denaturation (50 degrees C) of soluble uterine phosphodiesterase causes the loss of both cAMP and cGMP hydrolytic activities at the same rate. Isoelectric focusing reveals major (pI = 5.2) and minor forms (pI = 5.8) of phosphodiesterase which both catalyze the hydrolysis of the two cyclic nucleotide substrates. In vivo administration of estradiol produces identical decreases in the activities of cAMP and cGMP phosphodiesterase. These results raise the possibility that the uterus contains a single form of soluble phosphodiesterase which catalyzes the hydrolysis of both cAMP and cGMP.     

The identification and characterization of two cyclic nucleotide phosphodiesterases from bovine adrenal medulla

Two soluble cyclic nucleotide phosphodiesterase activities, designated Peak I (Mr = 216,000) and Peak II (Mr = 230,000), have been isolated from bovine adrenal medulla by DEAE-cellulose chromatography. Peak I has Ca2+-independent, cGMP-specific phosphodiesterase activity and Peak II has cGMP-stimulated cyclic nucleotide phosphodiesterase activity. Peak I hydrolyzes cGMP with hyperbolic kinetics and demonstrates a Km of 23 microM. Peak II hydrolyzes cGMP with hyperbolic kinetics but hydrolyzes cAMP with slightly sigmoidal kinetics and demonstrates Km values of 54 +/- 0.7 microM cGMP and 38 +/- 6 microM cAMP. Cyclic AMP and cGMP are competitive inhibitors of each other's hydrolysis, suggesting that these nucleotides may be hydrolyzed at the same catalytic site. Micromolar concentrations of cGMP cause a 5-fold stimulation of the hydrolysis of subsaturating concentrations of cAMP by the Peak II phosphodiesterase. Half-maximal activation occurs at 0.5 microM cGMP and the result of activation is a decrease in the apparent Km for cAMP. Stimulation of the hydrolysis of subsaturating concentrations of cGMP by cAMP was also detected; however, cAMP is a less potent activator of the enzyme than cGMP. Cyclic AMP causes a 1.5-fold stimulation of cGMP hydrolysis and half-maximal activation occurs at 2.5 microM cAMP.     

CYCLIC NUCLEOTIDE PHOSPHODIESTERASE OF HUMAN CEREBROSPINAL FLUID

Abstract— Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'–GMP (cGMP) phosphodiesterase activities were found in human cerebrospinal fluid (CSF) using low substrate concentration (0.4μM). More rapid hydrolysis of cGMP than that of cAMP was observed in human CSF. However, cGMP hydrolytic activity of CSF was very much lower (0.3 pmol/min/ml CSF) than that of human cerebral cortex (33.7 nmol/min/g wet cortex). The pH optimum was found to be 8.0 (cGMP phosphodiesterase) and 7.5 (cAMP phosphodiesterase). The maximum stimulation of both cAMP and cGMP phosphodiesterase was achieved at 4 mM-MgCl₂. Cyclic AMP had relatively little effect on the hydrolysis of cGMP in CSF and the cortex, while cGMP inhibited hydrolysis of cAMP in both tissues. Snake venom was found to stimulate cAMP and cGMP phosphodiesterase activity of CSF, by 60% and 110% respectively. This stimulation by snake venom was also observed in the cortex phosphodiesterase, but was not observed in human plasma or thyroid phosphodiesterase. When CSF was applied to Sepharose 6B column, cGMP phosphodiesterase was separated into three different molecular forms. A plot of activity against substrate concentration using peak I (largest molecular size) revealed a high affinity ( K _m= 2.6μM) and a low affinity ( K _m= 100μM) for cAMP suggesting the existence of at least two molecular forms of the enzyme. On the other hand, using a cGMP as substrate the only one K _m value (1.90 μm) was obtained. These K _m values of CSF enzymes described above were close to those obtained from human cerebral cortex preparations. The enzyme under peak I corresponded to the cortex enzyme when judged from its molecular size and stimulation by snake venom. It seems likely from our results that at least a part of CSF phosphodiesterase originates from the central nervous system.     

Bradykinin inhibition of cyclic AMP accumulation in D384 astrocytoma cells. Evidence against a role of cyclic GMP.

The present studies were performed in order to examine the possible role of cyclic GMP-stimulated phosphodiesterase (cGMP-PDE) activity in the inhibitory action of the inflammatory peptide bradykinin on cyclic AMP (cAMP) accumulation in D384 cells. Bradykinin decreased the forskolin-stimulated cAMP accumulation in the presence of the phosphodiesterase inhibitor rolipram, and caused a transient 50% rise in cellular cGMP in the presence of the nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). Both basal and bradykinin-stimulated cGMP accumulation were about 8 times higher in the presence of IBMX than in the presence of rolipram. Sodium nitroprusside, which caused a 20-70-fold increase in cGMP levels reduced forskolin stimulated cAMP accumulation, whereas hydroxylamine, which maximally caused a 16-fold increase in cGMP, did not. 8-bromo-cGMP or dibutyryl cGMP had no effect on cAMP accumulation induced by forskolin. The inhibitory effect of nitroprusside was totally reversed by blocking the soluble guanylate cyclase activity by methylene blue treatment; however, the inhibitory action of bradykinin on cAMP accumulation was not changed by this treatment. Additionally, inhibition of nitric oxide synthesis, which is known to be regulated by Ca2+ and in turn stimulates cGMP production, by N omega-nitro-L-arginine (L-NAME) treatment did not alter the inhibitory effect of bradykinin on forskolin-induced cAMP accumulation. These results indicate that large increases in cGMP may regulate cAMP via cGMP-PDE whereas the small increase induced by bradykinin is insufficient and that cGMP is not involved in the inhibitory action of bradykinin on cAMP levels in D384 cells.     

Cyclic nucleotide phosphodiesterases of rabbit renal cortex. Characterization of brush border membrane activities.

Cyclic nucleotide phosphodiesterase activity in brush border membranes, isolated from proximal tubule cells of the rabbit renal cortex, was investigated. Brush border cAMP phosphodiesterase activity was tightly bound to the membrane and was distinguished from the soluble phosphodiesterase activity of the renal cortex cytosol. Multiple forms of the brush border membrane cAMP phosphodiesterase activity, dependent on the concentration of substrate, were found. When assayed with 1 μm or 1 mm cAMP, activities differed in pH optimum, effects of various divalent cations, inhibition by metal ion chelators and reactivation by metals, thermolability, sensitivity to inhibitors and specificity.Renal brush border membranes also possessed cGMP phosphodiesterase activity. cAMP was a relatively poor inhibitor of the hydrolysis of 1 μm cGMP and the hydrolysis of 1 μm cAMP was virtually insensitive to cGMP. These findings suggest that the low substrate concentration-dependent cAMP phosphodiesterase was distinct from the low substrate concentration-dependent cGMP phosphodiesterase.Heat-stable effectors of phosphodiesterase activity were found in the renal cortex. One effector activated soluble cAMP phosphodiesterase. Activation was decreased by EGTA, enhanced by Ca²⁺ and diminished by preincubating the effector with proteolytic enzymes. The other heat-stable effector inhibited brush border membrane phosphodiesterase activity. Inhibition was unaffected by metal ions, unaffected by preincubating the effector with proteolytic enzymes, but diminished by preincubation with phospholipase C and neuraminidase.It is suggested that changes in the activity of the enzyme (or enzymes), which in turn controls, in part, the effective concentration of cAMP at its site (or sites) of action in the renal cell, may be significant in regulating hormonal-dependent transport in the proximal tubule.     

Aggregation states of cyclic nucleotide phosphodiesterase of murine thymocytes

In murine thymocytes cyclic nucleotide phosphodiesterase is represented by cAMP- and cGMP-specific forms. cAMP and cGMP phosphodiesterase activities showed anomalous kinetic behaviour indicative of 'low' and 'high' affinity enzyme forms. Sucrose density gradient centrifugation resolved only 'low' affinity forms of cAMP and cGMP phosphodiesterases. Gel filtration on Ultragel Aca 34 column showed that cAMP and cGMP phosphodiesterases are probably oligomeric enzymes. Storage of enzyme preparation at 4 degrees C for 24-48 h led to a decrease of higher molecular weight form and enhancement of cAMP and cGMP phosphodiesterase activities.     

Identification and characterization of two unusual cGMP-stimulated phoshodiesterases in dictyostelium

Recently, we recognized two genes, gbpA and gbpB, encoding putative cGMP-binding proteins with a Zn(2+)-hydrolase domain and two cyclic nucleotide binding domains. The Zn(2+)-hydrolase domains belong to the superfamily of beta-lactamases, also harboring a small family of class II phosphodiesterases from bacteria and lower eukaryotes. Gene inactivation and overexpression studies demonstrate that gbpA encodes the cGMP-stimulated cGMP-phosphodiesterase that was characterized biochemically previously and was shown to be involved in chemotaxis. cAMP neither activates nor is a substrate of GbpA. The gbpB gene is expressed mainly in the multicellular stage and seems to encode a dual specificity phosphodiesterase with preference for cAMP. The enzyme hydrolyses cAMP approximately 9-fold faster than cGMP and is activated by cAMP and cGMP with a K(A) value of approximately 0.7 and 2.3 microM, respectively. Cells with a deletion of the gbpB gene have increased basal and receptor stimulated cAMP levels and are sporogeneous. We propose that GbpA and GbpB hydrolyze the substrate in the Zn(2+)-hydrolase domain, whereas the cyclic nucleotide binding domains mediate activation. The human cGMP-stimulated cAMP/cGMP phosphodiesterase has similar biochemical properties, but a completely different topology: hydrolysis takes place by a class I catalytic domain and GAF domains mediate cGMP activation.     

Properties of multiple kinetic forms of soluble cyclic nucleotide phosphodiesterase activity of rat colonic mucosa

Soluble phosphodiesterase (EC 3.1.4.1) activity is 3-5-fold lower in superficial colonic epithelial cells compared to that in cells isolated from the lower colonic crypt. Higher phosphodiesterase activity in lower crypt cells is correlated with a 5-fold higher rate of incorporation of [3H]thymidine into DNA in these cells. DEAE-cellulose chromatography of the soluble fraction of superficial and proliferative colonic epithelial cells resulted in separation of three enzyme forms: (1) fraction I, an enzyme which hydrolyzes both cAMP and cGMP with high affinity (apparent Km cAMP = 5 +/- 1 microM, Km cGMP = 2.5 +/- 0.5 microM) and is stimulated 3-6-fold by Ca2+ plus calmodulin; (2) fraction II, a form which hydrolyzes both cAMP and cGMP with low affinity (S0.5 cAMP = 52 +/- 7 microM, S0.5 cGMP = 17 +/- 4 microM), exhibits positive copperativity with respect to substrate and shows cGMP stimulation of cAMP hydrolysis and (3) fraction III, a cAMP-specific form which exhibits biphasic kinetics, a low Km for cAMP (Km cAMP = 5 +/- 1 microM) and does not hydrolyze cGMP. The pattern of distribution of phosphodiesterase activities on DEAE-cellulose was similar in superficial and proliferative colonic epithelial cells. The higher specific activity in proliferative cells was reflected in higher activities of each of the three chromatographically distinct forms of the enzyme. In contrast to epithelial cells, the soluble fraction of homogenates of the submucosa and supporting cells exhibited phosphodiesterase forms I and II and was lacking in the form corresponding to fraction III of epithelial cells.     

cGMP-activated phosphodiesterase from human brain: kinetic and regulatory properties]

The kinetic and regulatory properties of cGMP-activated phosphodiesterase (PDE) from human brain were studied. In double reciprocal plots the enzyme activity is characterized by a linear dependence of cAMP and a nonlinear one for cGMP. Micromolar concentrations of cGMP accelerate cAMP hydrolysis (7-14-fold) with Ka for cGMP of 0.36 microM. Stimulation of cAMP hydrolysis is accompanied by a decrease of Km with no changes in Vmax. With a rise in the cGMP concentration above 5 microM PDE activation is changed by its inhibition. Both substrates act as competitive inhibitors towards each other. The Ki value for both cGMP and cAMP is 30 microM. After the increase in the cAMP (Bt)2 concentration the activation of 5 microM cAMP hydrolysis is accompanied by the enzyme inhibition. Both analogs competitively inhibit cGMP hydrolysis with Ki of 10 and 1500 microM for cGMP(Bt)2 and cAMP(Bt)2, respectively. The data obtained point to the existence of two binding sites for cyclic nucleotides, namely, a regulatory site which is highly specific for cGMP and a catalytic site responsible for the hydrolysis of the both substrates which displays no apparent specificity either for cAMP or for cGMP. The different affinity of natural and synthetic cyclic nucleotides for these sites is determined, to a large extent, by the amino groups in the 2nd and 6th positions of the purine ring.     

A Cytogenetic Analysis of Cyclic Nucleotide Phosphodiesterase Activities in Drosophila

The genome of Drosophila melanogaster has been surveyed for chromosomal regions which exert a dosage effect on the activities of cAMP phosphodiesterase or cGMP phosphodiesterase. Two regions increase cAMP phosphodiesterase activity when present as duplications. A region of the X chromosome increases cAMP phosphodiesterase activity when duplicated and decreases that activity when deficient. This region has been delimited to chromomeres 3D3 and 3D4, with 3D4 being the most probable locus, and may contain a structural gene for cAMP phosphodiesterase. A region on the third chromosome, 90E-91B, increases cAMP phosphodiesterase activity when duplicated but has no effect on the activity when deficient. Two regions increase cGMP phosphodiesterase activity when present as duplications. A region of the X chromosome, 5D-9C, increases cGMP phosphodiesterase activity when duplicated, but smaller duplications covering this region fail to show such an increase, indicating that a single locus is not responsible for the increase observed for the larger duplication. A region of the third chromosome, 88C-91B, also increases cGMP phosphodiesterase activity when duplicated. Smaller duplications covering this region show smaller increases than that observed for the larger duplication, suggesting that at least three loci between 88C and 91B contribute to the observed increase by that region. Deficiencies covering region 88C-91B do not affect cGMP phosphodiesterase activity. No locus for a presumptive structural gene for cGMP phosphodiesterase has been found. Limitations of the use of segmental aneuploidy in locating structural genes for enzymes are discussed.     

The role of cyclic nucleotides in the regulation of mitotic activity in SSPE virus-infected human brain tissue.

The intracellular level of cGMP was independent of the rate of cell division in cells derived from virally infected brain tissue. The phosphodiesterase inhibitor R07-2956 (4-dimethoxybenzyl-2-imidazolidinone) increased the intracellular level of cGMP in virally infected brain cells, but it did not effect the level of cAMP. There was no correction between the increase in cGMP levels following addition of R07-2956 and changes in mitotic activity in the brain cell cultures. Experimental manipulations which increased the cAMP level were accompanied by a decreased mitotic rate indicating there was a correlation between mitotic activity and the level of cAMP in the same cells. Raising the intracellular level of cAMP by exogenous db-cAMP or cAMP or the use of other phosphodiesterase inhibitors routinely increased the level of cGMP as well. Conversely increasing the intracellular cGMP level by adding the exogenous cGMP increased the level of both cGMP and cAMP.A tissue culture system was used with the cell line derived from viral infected human brain tissue originally obtained from a patient with subacute sclerosing panencephalitis (SSPE). The intracellular levels of cAMP and cGMP were monitored by radioimmunoassay following manipulation of the system by addition of exogenous cGMP (0.05 mM), addition of exogenous db-cAMP (0.5 mM), or cAMP (0.5 mM) and the use of phosphodiesterase inhibitors: theophylline (1.0 mM), papaverine (50 μg/ml), 4-3-butoxy-4-methoxy benzyl-2-imidozalidinone (R020-1724) and R07-2956. Cell division was monitored in treated and non-treated cultures at 24 h intervals by analyzing the cell number and mitotic index.High levels of cGMP were found in cells which were not actively dividing but high levels were just as apt to be present in dividing cells. There was an inverse relationship between cell division and the level of cAMP.     

Cyclic nucleotide metabolism in mouse epididymal spermatozoa during capacitation in vitro

The role of cyclic nucleotides in sperm capacitation is equivocal. Using conditions known to support mouse sperm capacitation after 120 min incubation in vitro, the cAMP and cGMP contents of epididymal spermatozoa were measured and the cGMP/cAMP ratio determined. The initial high cAMP content detected upon release of spermatozoa decreased within 30 min to a lower plateau, which was then maintained throughout incubation. With the cGMP content remaining approximately constant, the cGMP/cAMP ratio increased over 120 min. In the presence of 2 mM caffeine, an increased cAMP content was noted at 0 and 30 min before a fall to the plateau level. To investigate cyclic nucleotide metabolism, adenylate cyclase and phosphodiesterase activities were compared in two sperm populations, one essentially uncapacitated and the other incubated for 120 min. Adenylate cyclase activity, higher in the presence of 2 mM Mn²⁺ compared to Mg²⁺, showed increased activity at 120 min compared to 30 min incubation, while phosphodiesterase activity decreased during this period. The ability of spermatozoa to form adenosine and inosine from cAMP indicated endogenous 5′-nucleotidase and deaminase, as well as phosphodiesterase, activities. Although the endogenous cAMP content appeared to remain constant during the time that acrosome loss, hyperactivated motility and fertilizing ability can be demonstrated, activities of the enzymes responsible for cAMP metabolism indicate an increased potential for cAMP availability and turnover. The increased cGMP/cAMP ratio may also play a role during capacitation.     

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.     

The specificity of the cAMP receptor mediating activation of adenylate cyclase in Dictyostelium discoideum

In Dictyostelium discoideum amoebae, binding of cyclic AMP (cAMP) to surface receptors elicits numerous responses including chemotaxis, cyclic GMP (cGMP) accumulation, and activation of adenylate cyclase. The specificity of the surface cAMP receptor which mediates activation of adenylate cyclase and cAMP secretion was determined by testing the relative effectiveness of a series of 10 cAMP analogs. Each of the 10 analogs elicited cAMP secretion, chemotaxis, and cGMP accumulation in the same dose range. The order of potency for eliciting these responses (cAMP greater than 2'-H-cAMP greater than N1-O-cAMP greater than cAMPS(Sp) greater than 6-Cl-cAMP greater than cAMPN(CH3)2(Sp) greater than 3'-NH-cAMP greater than 8-Br-cAMP greater than cAMPS(Rp) greater than cAMPN(CH3)2(Rp] matches that for binding to the major cell surface cAMP binding sites and differs from that of the cell surface phosphodiesterase and the major intracellular cAMP binding protein.     

cGMP-dependent cyclic nucleotide phosphodiesterase from human lymphoid cells

The kinetic properties of cyclic nucleotide phosphodiesterase isolated from the cytoplasmic fraction of lymphoblastoma QOS cells were studied. It was demonstrated that the enzyme can be activated in the presence of micromolar concentrations of cGMP. The kinetic properties of the enzyme are characterized by the nonlinear dependence of the cAMP hydrolysis rate on the substrate concentration. The curve becomes linear in the presence of cGMP. The molecular mass of phosphodiesterase as determined from gel filtration data is 80,0000 Da.