Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

Plurality of cyclic nucleotide phosphodiesterase in Spinacea oleracea: subcellular distribution,partial purification,and properties

An active cyclic nucleotide phosphodiesterase has been partially purified from the 100 000 g supernatant of a spinach homogenate. It precipitated at 20–40% saturation with (NH₄)₂SO₄ and was separated on a column of Sephadex G-200 into two major peaks of activity (peaks 1 and 2). Peak 1 (MW 5 × 10⁵) was resolved by column chromatography on DEAE-cellulose into 5 protein fractions; two of these (1_c and 1_m) exhibited cyclic nucleotide phosphodiesterase activity. Subcellular fractionation showed that the phosphodiesterase of highest specific activity is located in the peroxisomes but that an enzyme of relatively high specific activity also occurs in the chloroplast and Golgi fractions. The largest total activity was in the microsomes. Isoelectric focussing of chloroplast phosphodiesterase activity gave two bands corresponding to peaks 1_c and 2. Similar examination of the microsomal, peroxisomal and Golgi fractions showed phosphodiesterases corresponding to peaks 1_m and 2. Peak 1_c activity is greater towards purine 3′,5′-cyclic nucleotides than towards their 2′,3′-isomers; the converse is true of peak 1_m. Examination of the properties of 1_c and 1_m showed a number of other differences. The pH optimum of 1_c is 6.1 and that of 1_m is 4.9. Theophylline (0.1 mM) inhibited 1_c to a greater extent than it did 1_m; Ca²⁺ stimulated 1_c activity but had no effect on 1_m. Pre-incubation with trypsin inhibited 1_m activity whereas similar treatment of 1_c gave an initial 5-fold stimulation. Repeated freezing and thawing of preparations 1_c and 1_m also evoked a difference in response. These results were shown to be attributable to removal of an inhibitor from 1_c. Evidence is presented that an endogenous activator is also present.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Multiple forms of cyclic nucleotide phosphodiesterase from bovine carotid artery smooth muscle.

DEAE-cellulose chromatography, in the presence and absence of Ca²⁺, of the 16,000g supernatant from bovine carotid artery smooth muscle has been used to separate four different types of cyclic nucleotide phosphodiesterase (3′:5′-cyclic-nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) activity, designated types A, B, C, and D. Type A is a high affinity, cyclic AMP-specific form of phosphodiesterase (K_m = 1.6 μM) and elutes at relatively high ionic strength. Type B is a high affinity (K_m = 2 μM), cyclic GMP-specific form which elutes at low ionic strength. Type C is a mixed substrate form, displaying anomalous kinetics for the hydrolysis of both cyclic AMP and cyclic GMP. It elutes from DEAE-cellulose at an ionic strength intermediate to that of types A and B. Type D is also a mixed substrate form of phosphodiesterase. However, its elution pattern from DEAE-cellulose differs, depending on whether Ca²⁺ is present or not, suggesting a Ca²⁺-dependent interaction between this enzyme form and the acidic Ca²⁺-dependent regulator protein (CDR). The hydrolytic activity of type D is stimulated by CDR, and activation requires the simultaneous presence of Ca²⁺ and CDR. Kinetic analysis of cyclic AMP hydrolysis by type D gives a linear double reciprocal plot; activation has no effect on the K_m but increases the velocity approximately sixfold. Activation of cyclic GMP hydrolysis apparently affects both the K_m and V. At all concentrations tested, the degree of activation is higher with cyclic AMP than with cyclic GMP. It is suggested that while the activable form of phosphodiesterase may play a relatively minor role in the overall hydrolysis of cyclic nucleotides, Ca²⁺-dependent activation may have a more important role in regulating the level of cyclic AMP than that of cyclic GMP in vascular smooth muscle.     

A further study on the regulation of cyclic nucleotide phosphodiesterase activity in neuroblastoma cells: Effect of growth

Summary Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) phosphodiesterase activity in mouse neuroblastoma cells in culture markedly increased during exponential growth and reached a maximal level at confluency; whereas guanosine 3′, 5′-cyclic monophosphate (cyclic GMP) phosphodiesterase activity only slightly but significantly increased under a similar experimental condition. The increase in cyclic AMP phosphodiesterase activity was blocked by both cycloheximide and dactinomycin, whereas the increase in cyclic GMP phosphodiesterase was blocked by only cycloheximide. When the confluent cells were replated at low density, the cyclic nucleotide phosphodiesterase activity decreased; however, when they were plated at high cell density which equaled confluency, the enzyme activity did not decrease. Unlike cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity did not change significantly in prostaglandin E₁-treated cells, but decreased in cells treated with the inhibitor of phosphodiesterase. Like cyclic AMP phosphodiesterase activity, cyclic GMP phosphodiesterase activity also did not change in cells treated with serum-free medium, X-irradiation, sodium butyrate and 6-thioguanine. This work was supported by USPHS NS-09230, and DRG-1273 from Damon Runyon-Walter Winchell Cancer Fund.     

Regulation by a β-adrenergic receptor of a Ca2+-independent adenosine 3′,5′-(cyclic)monophosphate phosphodiesterase in C6 glioma cells

The hormonal control of cyclic nucleotide phosphodiesterase (EC 3.1.4.17) activity has been studied by using as a model the isoproterenol stimulation of cyclic AMP phosphodiesterase activity in C6 glioma cells. A 2-fold increase in cyclic AMP phosphodiesterase specific activity was observed in homogenates of isoproterenol-treated cells relative to control. This increase reached a maximum 3 h after addition of isoproterenol, was selective for cyclic AMP hydrolysis, was reproduced by incubation with 8-Br cyclic AMP but not with 8-Br cyclic GMP and was limited to the soluble enzyme activity. The presence of 0.1 mM EGTA did not alter the magnitude of the increase in phosphodiesterase activity. Moreover, the calmodulin content in the cell extracts was not changed after isoproterernol. DEASE-Sephacel chromatography of the 100 000×g supernatant resolved two peaks of phosphodiesterase activity. The first peak hydrolyzed both cyclic nucleotides and was activated by Ca²⁺ and purified calmodulin. The second peak was specific for cyclic AMP but it was Ca²⁺- and calmodulin-insensitive. Isoproterenol selectively increased the specific activity of the second peak. Kinetic analysis of the cyclic AMP hydrolysis by the induced enzyme reveled a non-linear Hofstee plot with apparent K_m values of 2–5 μM. Cyclic GMP was not hydrolyzed by this enzyme in the absence or presence of calmodulin and failed to affect the kinetics of the hydrolysis of cyclic AMP. Gel filtration chromatography of the induced DEASE-Sephacel peak resolved a single peak of enzyme activity with an apparent molecular weight of 54 000.     

The role of adenosine 3':5'-cyclic monophosphate in relation to the diuretic hormone of Rhodnius prolixus.

The relationship between diuretic hormone (DH) and adenosine 3′:5′-cyclic monophosphate (cyclic AMP) in Rhodnius Malpighian tubules has been investigated. Direct measurement of cyclic AMP levels during stimulation of the tubules by DH supports the view that cyclic AMP is a ‘second messenger’ in this system.Also, the activity of endogenous cyclic AMP phosphodiesterase and its inhibition by theophylline has been investigated briefly. Certain other 3′:5′-cyclic nucleotides have been examined for diuretic activity on Rhodnius Malpighian tubules.     

Partial purification of adenosine 3',5'-cyclic monophosphate phosphodiesterases from rat pancreas in the presence of excess protease inhibitors.

(i) Three forms of cyclic AMP phosphodiesterases (3′,5′-cyclic AMP 5′-nucleotidohydrolase, EC 3.1.4.17), F1, F2-I and F2-II, were partially purified from the soluble fraction of rat pancreas in the presence of excess protease inhibitors by DEAE-cellulose column chromatography and gel filtration and were characterized. (ii) F2-II, which was purified 31-fold, exhibited a single peak of activity on both polyacrylamide-gel electrophoresis and isoelectric focusing. The enzyme had a molecular weight of about 70,000, an isoelectric point of 3.9, and an optimal pH around 8.5 and required Mg²⁺ or Mn²⁺ but not Ca²⁺ for activity. The K_m values of this enzyme for cyclic AMP and cyclic GMP were 1 and 50 μm, respectively, while V values of this enzyme for cyclic AMP and cyclic GMP were 36.1 and 12.6 nmol min^?1 (mg of protein)^?1, respectively. Cyclic GMP competitively inhibited hydrolysis of cyclic AMP by this enzyme. Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone] also inhibited hydrolysis of cyclic AMP competitively, with a K_i value of 1 μm. (iii) Fraction F1, which was purified 10-fold, had a molecular weight of more than 500,000 and required Mg²⁺ for activity. Its K_m values for cyclic AMP were 1 and 5 μm. Its K_m value for cyclic GMP was 45 μm. Fraction F2-I, which was purified 26-fold, had a molecular weight of about 70,000. The ratio of the initial velocity of hydrolysis of cyclic GMP to that of cyclic AMP was 0.5 at a substrate concentration of 1 μm.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Purine Nucleoside 3′,5′-Cyclic Monophosphates as Hormonal Modulators of Cellular Proliferation,Metastases and Lymphocyte Response

Abstract

A review of the potential role of cAMP and cGMP as hormonal regulators of tumor cell proliferation, metastases and lymphocyte activation reveals that several synthetic purine nucleoside 3′,5′-cyclic monophosphates are more potent and more selective in modulating certain specific responses than the parent natural cyclic nucleotides. cAMP derivatives have been prepared which will temporarily restore transformed cells to the normal phenotype. cAMP analogs may well be found which will selectively inhibit tumor metastases. Certain cGMP analogs could selectively stimulate the lymphocyte response toward the destruction of tumor cells. The synthesis of new cyclic nucleotides should provide unique nontoxic agents that could combat neoplasia on a hormonal basis.     

The adnosine 3',5'-monophosphate and guanosine 3',5'-monophosphate phosphodiesterases from human lung tissue.

Human lung tissue contains phosphodiesterase enzymes capable of hydrolyzing both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP). The cyclic AMP enzyme exhibits three distinct binding affinities for its substrate (apparent Km = 0.4μM, 3μM, and 40μM) while the cyclic GMP enzyme reveals only two affinities (Km = 5μM and 40μM). The pH optima for the cyclic AMP and cyclic GMP phosphodiesterase are similar (pH 7.6–7.8). Both are inhibited by known inhibitors of phosphodiesterase activity (aminophylline, caffeine, and 3-isobutyl-1-methylxanthine). The divalent cations Mg²⁺ and Mn²⁺ stimulate cyclic AMP phosphodiesterase activity (in the absence of Mg²⁺) while Ca²⁺, Ni²⁺, and Cu²⁺ inhibit the enzyme. Histamine and imidazole slightly stimulate cyclic AMP hydrolytic activity. Thus, human lung tissue does contain multiple forms of both the cyclic AMP and cyclic GMP phosphodiesterase which are influenced by a variety of effectors.     

Improved synthesis of 32P-labelled 3′,5′-cyclic AMP, 3′,5′-cyclic GMP and other 3′,5′cyclic ribo- and deoxyribonucletodies of high specific activity

A general procedure is described for the two-step chemical synthesis from [³²P]orthophosphoric acid of the eight common ribo- and deoxyribonucleoside 3′,5′-cyclic monophosphates. The method is simple and reliable and both steps are carried out in the same reaction flask without an intermediate purification step. ³²P-labelled cyclic nucleotides are obtained after paper chromatography in yields of 20–60% relative to starting [³²P]orthophosphoric acid and with a specific activity of greater than 1 mCi/μmole. Alternative methods for the purification of reaction mixtures and for the preparation of ³²P-labelled 3′,5′-cyclic AMP and 3,′,5′-cyclic GMP are described.     

Characterization of particulate cyclic nucleotide phosphodiesterases of rat kidney.

Particulate cyclic nucleotide phosphodiesterases of rat kidney display some distinct kinetic and regulatory properties. Only a small portion (5–10%) of the total homogenate low K_m cyclic AMP phosphodiesterase activity (measured with concentrations of cyclic AMP less than l μm) is tightly associated with kidney membranes. Cyclic GMP phosphodiesterase activity (measured with 0.25–200 μm cyclic GMP) is readily detectable in these fractionated and washed membranes. Low concentrations of cyclic GMP stimulated the hydrolysis of cyclic AMP (K_a ～- 0.5 μM), an effect not noted in most other membrane systems. High concentrations of cyclic GMP (K_i ～- 450 μM) and cyclic AMP (K_i ～- 150 μM) inhibited the hydrolysis of each other noncompetitively. Solubilization of membrane bound activities by sonication or Sarkosyl L markedly alters enzyme kinetic properties and the responses to cyclic nucleotides and sulfhydryl reagents. Incubation of membrane fractions with dithiothreitol (5 mm) or storage of the membranes at 4 °C results in a change in extrapolated kinetic constants for cyclic AMP hydrolysis and an increase in the rate of denaturation at 45 °C. Our findings raise the possibility that regulation of membrane-bound cyclic nucleotide phosphodiesterase activity involves interactions with cyclic nucleotides themselves, as well as oxidation and reduction of disulfide bonds and membrane-enzyme interactions.     

Cartilage cyclic nucleotide phosphodiesterase: inhibition by anti-inflammatory agents

Soluble 3′,5′-nucleotide phosphodiesterase (PDE) activity is described in chicken epiphyseal and articular cartilage. Kinetic studies of these enzymes demonstrate a high and low K_m for the substrates, adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and guanosine 3′,5′-cyclic monophosphate (cyclic GMP). Epiphyseal and articular PDE activities are inhibited by those anti-inflammatory agents which are potent inhibitors of the enzyme, prostaglandin synthetase (PS). Specificity of this inhibition is indicated by the activity of these agents against the low K_m enzyme. Other anti-inflammatory agents with significantly less potency as PS inhibitors or with no activity against prostaglandin synthetase are found to be either inactive or relatively less potent as inhibitors of cartilage PDE activity. A variety of other anti-inflammatory or anti-rheumatic agents, which are not known to affect prostaglandin synthetase activity, are poor inhibitors of cartilage PDE activity. These data provide insight into the mechanism of action of certain anti-inflammatory agents and into the relationships between prostaglandins and inflammatory reactions.     

REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASES OF CEREBRAL CORTEX BY Ca2+ AND CYCLIC GMP

Cyclic nucleotide phosphodiesterase activity of porcine cerebral cortical extracts was measured with 0.1–100 μM-cyclic AMP and cyclic GMP and found to be dependent on both Ca²⁺ and added cyclic nucleotides. With decreasing substrate concentration activity with cyclic GMP became more dependent on Ca²⁺ whereas hydrolysis of cyclic AMP became less dependent. Cyclic GMP at 3 μM stimulated the hydrolysis of 0.1–10μM-cyclic AMP in the absence of Ca²⁺ (< 10^-10M) but inhibited activity with 200 μM-Ca²⁺ present. This differential, substrate- and Ca²⁺-dependent regulation was attributed to the presence of at least two types of phosphodiesterase distinguishable by DEAE-column chromatography. In the absence of Ca²⁺, activity with 1 μM-cyclic GMP eluted in one minor peak followed by two major peaks, D-I and D-II. Activity with 1 μM-cyclic AMP eluted almost entirely in D-II. Hydrolysis of cyclic AMP in D-II was activated by cyclic GMP. With added Ca²⁺ plus a Ca²⁺-dependent regulator (CDR), activity with 1 μM-cyclic GMP was markedly increased and eluted entirely at D-I. Total activity with 1 μM-cyclic AMP was only moderately increased and eluted as D-I with a shoulder at D-II. Elution profiles with 100 μM-substrate were relatively independent of substrate, with D-I predominant with Ca²⁺·CDR present and D-II predominant in its absence. Kinetic analysis of rechromatographed D-I showed a 20- to 40-fold activation by Ca²⁺·CDR that was largely due to an increase in V_max, with only 50% decreases in K_m Both substrates competitively inhibited hydrolysis of the other with K_i values equal to their respective K_m values (1.7 μM for cyclic GMP and 48 μM for cyclic AMP with Ca²⁺-CDR present). Studies with theophylline and trifluoperazine indicate differential, substrate-dependent inhibitions of both enzymes. These findings demonstrate that phosphodiesterase activity in neural tissue is subject to regulation by Ca²⁺, cyclic GMP, and inhibitors in a complex, substrate-specific and concentration-dependent manner.     

Adenylate cyclase and cyclic nucleotide phosphodiesterases in the developing rat liver

A potential regulatory role for the cyclic nucleotides during liver morphogenesis will be better understood as the development of various components of the cyclic nucleotide system are characterized. Accordingly, adenylate cyclase response to glucagon and 5′-guanylimidodiphosphate (Gpp(NH)p) and the specific activities, cellular distributions, and kinetic constants (V and K_m) of the cyclic AMP and cyclic GMP phosphodiesterases were determined at variuos stages of rat liver development. These results show (1) a period of increasing sensitivity of rat liver adenylate cyclase to glucagon at a time when sensitivity to NaF and Gpp(NH)p remains unchanged, and (2) increased responsiveness to glucagon plus Gpp(NH)p which is dependent upon the degree of glucagon sensitivity. It is concluded that the guanul nucleotide regulatory site is a functional part of adenylate cyclase very early in liver development and that the development of glucagon sensitivity is more probably limited by the developmet of glucagon receptors. Two forms of each phosphodiesterase (high and low K_m) were found throughout, except that low K_m cyclic GMP phosphodiesterase could not be demonstrated in the embryo. No significant change with age was found for the K_m or V of any of the enzyme forms. The ratio of soluble: particulate cyclic AMP phosphodiesterase decreased with age, whereas no change in the ration for cyclic GMP phosphodiesterase was observed. Specific activities of each enzyme from were highest in the perinatal period and decreased with age. The changes in phosphodiesterase specific activities paralled changes in guanylate and adenylate cyclase activities, which argues against a selective regulatory role for phosphodiesterase in modulating cyclic nucleotide influences during liver morphogenesis.     

ADRENAL MEDULLARY CYCLIC NUCLEOTIDE PHOSPHODIESTERASE.—REGULATORY PROPERTIES OF THREE ENZYME ACTIVITIES

Abstract— Cyclic nucleotide phosphodiesterase from bovine adrenal medulla was fractionated into multiple activities by two different procedures, sucrose gradient centrifugation and gel filtration. Extracts of frozen and thawed adrenal medulla homogenates gave two phosphodiesterase activity peaks following density gradient centrifugation. The higher molecular weight activity hydrolyzed both cyclic AMP and cyclic GMP; ethylene glycol-bis(aminoethyl ether)- N,N' -tetraacetic acid (EGTA) inhibited only the hydrolysis of cyclic GMP. The lower molecular weight activity hydrolyzed only cyclic AMP and was not inhibited by EGTA. The two activities were not interconverted by recentrifugation.
Gel filtration of cyclic nucleotide phosphodiesterase activity extracted from frozen and thawed adrenal medulla on Ultrogel AcA 34 resolved the enzyme into three distinct peaks of enzyme activity with molecular weights of 350,000 (Peak I), 229,000 (Peak II) and 162,000 (Peak III). The enzyme from fresh tissue was resolved into peak I and II and only a small fraction of Peak III. Peak I hydrolyzed both cyclic nucleotides, while peak II was a cyclic GMP-specific enzyme and peak III was specific for cyclic AMP. The hydrolysis of cyclic AMP by the activity in Peak I was markedly stimulated by cyclic GMP; the hydrolysis of cyclic GMP by peak II was inhibited by EGTA and stimulated by calcium and CDR (calcium-dependent regulator protein). Peak III, which appears to be particulate, is not activated by either cyclic GMP or calcium and CDR.     

Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Synthesis of Pyrazolo[3, 4-d]Pyrimidine Ribonucleoside 3′, 5′-Cyclic Phosphates Related to cAMP,cIMP and cGMP1

Abstract

The synthesis of pyrazolo[3,4-d]pyrimidine ribonucleoside 3′, 5′-cyclic phosphates related to cAMP, cIMP and cGMP has been achieved for the first time. Phosphorylation of 4-amino-6-methylthio-1-β-D-ribo-furanosylpyrazolo[3,4-d]pyrimidine (1) with POCl₃ in trimethyl phosphate gave the corresponding 5′-phosphate (2a). DCC mediated intramolecular cyclization of 2a gave the corresponding 3′, 5′-cyclic phosphate (3a), which on subsequent dethiation provided the cAMP analog 4-amino-1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidine 3′, 5′-cyclic phosphate (3b). A similar phosphorylation of 6-methylthio-1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidin-4(5H)-one (5), followed by cyclization with DCC gave the 3′, 5′-cyclic phosphate of 5 (9a). Dethiation of 9a with Raney nickel gave the cIMP analog 1-β-D-ribofuranosylpyrazolo[3, 4-d]pyrimidin-4(5H)-one 3′, 5′-cyclic phosphate (9b). Oxidation of 9a with m-chloroperoxy benzoic acid, followed by ammonolysis provided the cGMP analog 6-amino-1-β-D-ribofuranosylpyrazolo [3, 4-d] pyrimidin-4(5H)-one 3′, 5′-cyclic phosphate (7). The structural assignment of these cyclic nucleotides was made by UV and H NMR spectroscopic studies.     

Human blood platelet 3': 5'-cyclic nucleotide phosphodiesterase. Isolation of low-Km and high-Km phosphodiesterase.

Human blood platelet contained at least three kinetically distinct forms of 3': 5'-cyclic nucleotide phosphodiesterase (3': 5'-cyclic-AMP 5'-nucleotidohydrolase, EC 3.1.4.17) (F I, F II, and F III) which were clearly separated by DEAE-cellulose column chromatography. Although a few properties of the platelet phosphodiesterases such as their substrate affinities and DEAE-cellulose profile resembled somewhat those of the three 3': 5'-cyclic nucleotide phosphodiesterase in rat liver reported by Russell et al. [10], there were pronounced differences in some properties between the platelet and the liver enzymes: (1) the platelet enzymes hydrolyzed both cyclic nucleotides and lacked a highly specific cyclic guanosine 3': 5'-monophosphate (cyclic GMP) phosphodiesterase and (2) kinetic data of the platelet enzymes indicated that cyclic adenosine 3': 5'-monophosphate (cyclic AMP) and cyclic GMP interact with a single catalytic site on the enzyme. F I was a cyclic nucleotide phosphodiesterase with a high Km for cyclic AMP and a negatively cooperative low Km for cyclic GMP. F II hydrolyzed cyclic AMP and cyclic GMP about equally with a high Km for both substrates. F III was low Km phosphodiesterase which hydrolyzed cyclic AMP faster than cyclic GMP. Each cyclic nucleotide acted as a competitive inhibitor of the hydrolysis of the other nucleotide by these three fractions with Ki values similar to the Km values for each nucleotide suggesting that the hydrolysis of both cyclic AMP and cyclic GMP was catalyzed by a single catalytic site on the enzyme. However, cyclic GMP at low concentration (below 10 muM) was an activator of cyclic AMP hydrolysis by F I. Papaverine and EG 626 acted as competitive inhibitors of each fraction with virtually the same Ki value in both assays using either cyclic AMP or cyclic GMP as the substrate. The ratio of cyclic AMP hydrolysis to cyclic GMP hydrolysis by each fraction did not vary significantly after freezing/thawing or heat treatment. These facts also suggest that both nucleotides were hydrolyzed by the same catalytic site on the enzyme. The differences in apparent Ki values for inhibitors such as cyclic nucleotides, papaverine and EG 626 would indicate that three enzymes were different from each other. Centrifugation in a continuous sucrose gradient revealed sedimentation coefficients F I and II had 8.9 S and F III 4.6 S. The molecular weight of these forms, determined by gel filtration on a Sepharose 6B column, were approx. 240 000 (F I and II) and 180 000 (F III). F III was purified extensively (70-fold) from homogenate, with a recovery of approximately 7%.