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.     

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.     

Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.

Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.     

Cyclic nucleotide phosphodiesterases in somatic and germ cells of mouse seminiferous tubules

The distribution of phosphodiesterase forms in somatic and germ cells, and their variations during testicular development and germ cell differentiation have been investigated. Seminiferous tubules from immature mice and Sertoli cells in culture possessed two enzyme activities which were comparable to forms described for different tissues and species: (a) a calcium-calmodulin-dependent enzyme with high affinity for guanosine 3',5'-(cyclic)-monophosphate (cGMP), and (b) a calcium-calmodulin-independent enzyme with high affinity for adenosine 3',5'-(cyclic)-monophosphate (cAMP) the activity of which increased in cultured Sertoli cells after treatment with FSH or dibutyryl cAMP. Seminiferous tubules from adult animals and germ cells at the meiotic and post-meiotic stage of differentiation possessed two enzyme forms that could be distinguished from those present in somatic cells of the seminiferous tubules: (a) a calcium-calmodulin-dependent form with high affinity for both cAMP and cGMP, similar to forms described in other tissues from different species, and (b) a calcium-calmodulin-independent phosphodiesterase with high affinity for cAMP and present only in post-meiotic cells, previously identified also in germ cells of the rat.     

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.     

Rat sperm enzymes during epididymal transit

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).     

Isolation, characterization and Ca2+ and calmodulin regulation of cyclic nucleotide phosphodiesterases from human placentae of different ages

Two kinds of phosphodiesterases were isolated from human placenta by DEAE chromatography and characterized: one Ca2+ and calmodulin dependent, the other stimulated by Ca2+ but not by calmodulin. Both hydrolyzed cAMP and cGMP. The first one exhibited a higher affinity for cGMP. Half maximal activation by calmodulin was attained at 10(-8)M of calmodulin concentration independently of the hydrolyzed substrate (cGMP or cAMP). This phosphodiesterase appears to be almost homogeneous by molecular sieve chromatography on Ultragel AcA 34. The second phosphodiesterase exhibited similar affinities for cAMP and cGMP and could be resolved into three active isoforms with different molecular weight on Ultrogel AcA 34. Only minor differences were observed in the characteristics of these enzymes when the phosphodiesterases were prepared from placentae of 7-8 weeks of pregnancy or from normal term placenta.     

The cyclic adenosine monophosphate phosphodiesterases of myoblasts, fibroblasts, and their somatic cell hybrids.

Three forms of cAMP phosphodiesterases are found in mouse L cells (fibroblasts) and rat skeletal myoblasts. The myoblast enzymes can be resolved by chromatography on DEAE-cellulose and the fibroblast enzymes by chromatography on DEAE-Biogel. The myoblast enzymes are "high affinity" cAMP specific forms and have different molecular weights, while all L-cell enzymes have an apparent molecular weight of 450,000. Only one of the L-cell enzymes is able to hydrolyze both cyclic guanosine monophosphate (cGMP) and cAMP. Hydrolysis of the latter is stimulated by micromolar amounts of cGMP. The myoblast x L cell hybrids possess at least five phosphodiesterases, three of which can be identified as being of myoblast or fibroblast origin. One of the fibroblast enzymes appears to be modified in hybrids. The entire phosphodiesterase regulatory system of the myoblasts is active in the hybrids.     

Characterization of phosphodiesterase catalytic sites by means of cyclic nucleotide derivatives

Cyclic nucleotide derivatives have been used as a tool to characterize distinct catalytic sites on phosphodiesterase enzyme forms: the cGMP-stimulated enzyme from rat liver and the calmodulin-sensitive enzyme from rat or bovine brain. Under appropriate assay conditions, the analogues showed linear competitive inhibition with respect to cAMP (adenosine 3',5'-monophosphate) as substrate. The inhibition sequence of the fully activated cGMP-stimulated phosphodiesterase was identical to the inhibition sequence of the desensitized enzyme, i.e. the enzyme which has lost its ability to be stimulated by cGMP. The inhibition pattern could, therefore, not be attributed to competition with cGMP at an allosteric-activating site. Also, the inhibition sequence of the calmodulin-sensitive phosphodiesterase was maintained whether activity was basal or fully stimulated by calmodulin. When cAMP and cGMP, with identical chemical ligands substituted at the same position, were compared as inhibitors of the calmodulin-sensitive phosphodiesterase, the cGMP analogues were always the more potent suggesting that, for that enzyme, the catalytic site was sensitive to a guanine-type cyclic nucleotide structure. Comparing the two phosphodiesterases, it was possible to establish both similar and specific inhibitor potencies of cyclic nucleotide derivatives. In particular, the two enzymes exhibited large differences in analogue specificity modified at C-6, 6-chloropurine 3',5'-monophosphate or purine 3',5'-monophosphate.     

Cell-cycle-specific activity of cyclic nucleotide phosphodiesterase in Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. The activities of plasmodial homogenate and of selected subcellular fractions were measured. The results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. The whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 X 10(-4) M cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. The Km values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Substrate and effector specificity of a guanosine 3':5'-monophosphate phosphodiesterase from rat liver.

Guanosine 3':5'-monophosphate phosphodiesterases, which appear to be under allosteric control, have been partially purified from rat liver supernatant and particulate fractions. The preferred substrate for both phosphodiesterases was cGMP (Km values: cGMP less than cIMP less than cAMP). At subsaturating concentrations of substrate, the phosphodiesterases were stimulated by purine cyclic nucleotides. The order of effectiveness for activation of cyclic nucleotide hydrolysis was cGMP greater than cIMP greater than cAMP greater than cXMP. Using cAMP derivatives as activators of cIMP hydrolysis, modifications in the ribose, cyclic phosphate, and purine moieties were shown to alter the ability of the cyclic nucleotide to activate the supernatant enzyme. cGMP, at concentrations that stimulated cyclic nucleotide hydrolysis, enhanced chymotryptic inactivation of the supernatant phosphodiesterase. At similar concentrations, cAMP was not effective. It appears that on interaction with appropriate cyclic nucleotides, this phosphodiesterase undergoes conformational changes that are associated with increased catalytic activity and enhanced susceptibility to proteolytic attack. Divalent cation may not be required for the nucleotide-phosphodiesterase interaction and resultant change in conformation.     

An intracellular modulator of the 3'5'-cGMP hydrolysis in growing Dictyostelium discoideum amoebae

A heat-stable and acid-stable macromolecular factor present in the cytosol of growing Dictyostelium discoideum amoebae affects specifically the intracellular cGMP phosphodiesterase. It decreases the V of the enzyme but does not alter its Km. It has no effect on the cAMP or cGMP hydrolysis catalyzed by the intracellular cAMP-cGMP phosphodiesterases or by the extracellular phosphodiesterase. It is also expressed in a mutant (HPX235), defective in the synthesis of the cAMP-cGMP phosphodiesterases but capable of intracellular transduction of the chemotactic signal. This factor is resistant to several nucleases, proteases and phospholipases, and has an apparent molecular weight between 3500-10000. In contrast, the protein phosphodiesterase inhibitor secreted by the amoebae exerts an opposite inhibition on the intracellular phosphodiesterases. These two inhibitory factors may regulate intracellular cGMP hydrolysis during the chemotactic response.     

A new cGMP phosphodiesterase isolated from bovine platelets is substrate for cAMP- and cGMP-dependent protein kinases: evidence for a key role in the process of platelet activation.

The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate a specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion-exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a "pseudo-site" when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 mumol/min/mg. Moreover, this enzyme was phosphorylated by cAMP- and cGMP-dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases.     

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.     

Purification of the putative hormone-sensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity ligand

A "low Km" cAMP phosphodiesterase with properties of a peripheral membrane protein accounts for approximately 90% of total cAMP phosphodiesterase activity in particulate (100,000 X g) fractions from rat fat cells. Incubation of fat cells with insulin for 10 min increased particulate (but not soluble) cAMP phosphodiesterase activity, with a maximum increase (approximately 100%) at 1 nM insulin. Most of the increase in activity was retained after solubilization (with non-ionic detergent and NaBr) and partial purification (approximately 20-fold) on DEAE-Sephacel. The solubilized enzyme from adipose tissue was purified approximately 65,000-fold to apparent homogeneity (yield approximately 20%) by chromatography on DEAE-Sephacel and Sephadex G-200 and affinity chromatography on aminoethyl agarose conjugated with the N-(2-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamide (OPC 3689). A 63,800 +/- 200-Da polypeptide (accounting for greater than 90% of the protein eluted from the affinity column) was identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (with or without reduction). Enzyme activity was associated with the single protein band after electrophoresis under nondenaturing conditions. On gel permeation, Mr(app) was 100,000-110,000, suggesting that the holoenzyme is a dimer. A pI of 4.9-5.0 was estimated by isoelectric focusing. At 30 degrees C, the purified enzyme hydrolyzed both cAMP and cGMP with normal Michaelis-Menten kinetics; the pH optimum was 7.5. The Km(app) for cAMP was 0.38 microM and Vmax, 8.5 mumol/min/mg; for cGMP, Km(app) was 0.28 microM and Vmax, 2.0 mumol/min/mg. cGMP competitively inhibited cAMP hydrolysis with a Ki of approximately 0.15 microM. The enzyme was also inhibited by several OPC derivatives and "cardiotonic" drugs, but not by RO 20-1724. It was very sensitive to inhibition by agents which covalently modify protein sulfhydryls, but not by diisopropyl fluorophosphate. The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate low Km cAMP phosphodiesterase activity of rat adipocytes.     

Cell-Cycle-Specific Activity of Cyclic Nucleotide Phosphodiesterase In Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. the activities of plasmodial homogenate and of selected subcellular fractions were measured. the results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. the whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 × 10^-4 m cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. the K_m values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Cyclid 3':5'-nucleotide phosphodiesterase. Interconvertible multiple forms and their effects on enzyme activity and kinetics.

An extract of rat liver or human platelet displayed three cyclic 3':5'-nucleotide phosphodiesterase activity peaks (I, II, and III) in a continuous sucrose density gradient when assayed with millimolar adenosine 3':5'-monophosphate (cAMP) or guanosine 3':5'-monophosphate (cGMP). The three fractions obtained from each nucleotide were not superimposable. The molecular weights corresponding to the three activity peaks of cAMP phosphodiesterase in rat liver were approximately: I, 22,000; II, 75,000; and III, 140,000. In both tissues, fraction I was barely detectable when assayed with micromolar concentrations of either nucleotide, presumably because fraction I has low affinity for cAMP and cGMP. Any one of the three forms upon recentrifugation on the gradient generated the others, indicating that they were interconvertible. The multiple forms appear to represent different aggregated states of the enzyme. The ratio of the three forms of cAMP phosphodiesterase in the platelet was shifted by dibutyryl cAMP (B2cAMP) and by the enzyme concentration. B2cAMP enhanced the formation of fraction I. Low enzyme concentration favored the equilibrium towards fraction I, while high enzyme concentration favored fraction III. When phosphodiesterase activities in the extract of rat liver, human platelets, or bovine brain were examined as a function of enzyme concentration, rectilinear rates were observed with micromolar, but not with millimolar cAMP or cGMP. The specific activity with millimolar cAMP was higher with low than with high protein concentrations, suggesting that the dissociated form catalyzed the hydrolysis of cAMP faster than that of the associated form. In contrast, the specific activity with millimolar cGMP was lower with low than with high protein concentrations. Supplementing the reaction mixture with bovine serum albumin to a final constant protein concentration did not affect the activity, suggesting that the concentration of the enzyme rather than that of extraneous proteins affected the enzyme activity. A change in enzyme concentration affected the kinetic properties of phosphodiesterase. A low enzyme concentration of cAMP phosphodiesterase yielded a linear Lineweaver-Burk plot, and a Km of 1.2 X 10(-4) M (bovine), 3 X 10(-5) M (platelet), or 5 X 10(-4) M (liver), while a high enzyme concentration yielded a nonlinear plot, and apparent Km values of 1.4 X 10(-4) M and 2 X 10(-5) M (brain), 4 X 10(-5) M and 3 X 10(-6) M (platelet), or 4 X 10(-5) M and 3 X 10(-6) (liver). Since a low enzyme concentration favored fraction I, the dissociated form, whereas a high enzyme concentration favored fraction III, the associated form, these kinetic constants suggest that the dissociated form exhibits a high Km and the associated form exhibits a low Km. In contrast, a high enzyme concentration gave a linear kinetic plot for cGMP phosphodiesterase, while a low enzyme concentration gave a nonlinear plot...     

Calmodulin-stimulated cyclic nucleotide phosphodiesterases in plasma membranes of bovine epididymal spermatozoa

Cyclic nucleotide phosphodiesterase in the plasma membranes of bovine epididymal spermatozoa was stimulated by added Ca2+ and calmodulin. The rate of hydrolysis and responsiveness toward calmodulin was greater for cAMP than for cGMP. The kinetic analysis of the activity revealed two forms of phosphodiesterase with apparent Km values of 7.5 and 95 microM for cAMP. Calmodulin stimulated both of the activities by increasing the Vmax without affecting the Km's. The activity response with respect to Ca2+ concentration appears to be biphasic in both the absence and presence of added calmodulin. Trifluoperazine inhibited the Ca2+- and calmodulin-sensitive enzyme activity in a dose-dependent manner. The calmodulin-stimulated phosphodiesterase activity in the sperm plasma membranes can be solubilized and absorbed to a Calmodulin-Sepharose affinity column in the presence of Ca2+.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca²⁺-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.     

Cardiac cyclic nucleotide phosphodiesterases: function, regulation, and therapeutic prospects

The second messengers cAMP and cGMP exist in multiple discrete compartments and regulate a variety of biological processes in the heart. The cyclic nucleotide phosphodiesterases, by catalyzing the hydrolysis of cAMP and cGMP, play crucial roles in controlling the amplitude, duration, and compartmentalization of cyclic nucleotide signaling. Over 60 phosphodiesterase isoforms, grouped into 11 families, have been discovered to date. In the heart, both cAMP- and cGMP-hydrolyzing phosphodiesterases play important roles in physiology and pathology. At least 7 of the 11 phosphodiesterase family members appear to be expressed in the myocardium, and evidence supports phosphodiesterase involvement in regulation of many processes important for normal cardiac function including pacemaking and contractility, as well as many pathological processes including remodeling and myocyte apoptosis. Pharmacological inhibitors for a number of phosphodiesterase families have also been used clinically or preclinically to treat several types of cardiovascular disease. In addition, phosphodiesterase inhibitors are also being considered for treatment of many forms of disease outside the cardiovascular system, raising the possibility of cardiovascular side effects of such agents. This review will discuss the roles of phosphodiesterases in the heart, in terms of expression patterns, regulation, and involvement in physiological and pathological functions. Additionally, the cardiac effects of various phosphodiesterase inhibitors, both potentially beneficial and detrimental, will be discussed.