Properties of cyclic nucleotide phosphodiesterase of the rabbit myometrium in various functional states

Chromatography on DEAE-cellulose of a soluble sulfate-precipitated fraction of cyclic nucleotide phosphodiesterase from rabbit myometrium revealed two 3':5'-GMP and 3':5'-AMP-hydrolase activities. 3':5'-GMP phosphodiesterase (fraction I) was eluted with 0.15-0.23 M NaCl, while 3':5'-AMP phosphodiesterase (fraction II) with 0.2-0.35 M NaCl. 3':5'-GMP phosphodiesterase hydrolyzed 3':5'-GMP with Km = 14 microM and V = 5.25 nmol . min . mg of protein, while 3':5'-AMP phosphodiesterase hydrolyzed both cyclic nucleotides with Km for 3':5'-GMP equal to 12 microM and V = 1.33 nmol . min . mg of protein; the Km value for 3':5'-AMP was 3.6 and 30.5 microM, respectively; the corresponding values of V were 0.28 and 0.97 nmol . min . mg of protein. In late pregnancy, the level of the 3':5'-AMP hydrolase activity of rabbit myometrium was significantly elevated in parallel with an increase in V, predominantly for the enzyme with a low affinity for 3':5'-AMP. The 3':5'-GMP hydrolase activity and V were largely decreased for both phosphodiesterase fractions; the Km value for fraction I was also diminished. During labour, the rate of 3':5'-AMP hydrolysis by myometrium phosphodiesterase was decreased down to the level typical of functional rest. The rate of 3':5'-GMP hydrolysis during the same period by fraction I remained at a low level, i. e., as in pregnancy, while that of fraction II was increased up to the level typical of functional rest.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the cyclic 3',5'-nucleotide phosphodiesterase actitivty associated with synaptosomal plasma membranes and synaptic junctions.

Some characteristics of the cyclic 3',5'-nucleotide phosphodiesterase (phosphodiesterase) activity associated with the synaptosomal plasma membrane (synaptic membrane) and the synaptic junction fractions of rat brain are reported. Kinetic analysis revealed that only one type of phosphodiesterase activity, with a Km of 2.10 19(-4) M for cyclic AMP, is associated with both fractions. The specific activities of the phosphodiesterase in synaptic membranes and synaptic junctions have been estimated at 23.4 nmol/min per mg protein and 22.5 nmol/min per mg protein, respectively. The synaptic junction-associated activity undergoes a 30% stimulation by Ca2+ while no Ca2+ sensitivity of the synaptic membrane-associated activity could be detected. Cytochemical studies performed on the synaptic membrane fraction demonstrated a predominant localization of phosphodiesterase activity over postsynaptic densities, while dense deposits were sometimes observed over the synaptic cleft region.     

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...     

Cyclic nucleotide phosphodiesterase. Partial purification and characterization of a high affinity enzyme activity from human lung tissue.

Part of the soluble cyclic nucleotide phosphodiesterase activity of crude human lung tissue can be attributed to a thermosensitive (37 degrees) enzyme with a high apparent affinity for both adenosine 3':5'-monophosphate (cyclic AMP) and guanosine 3':5'-monophosphate (cyclic GMP). The enzyme can be partially purified by DEAE-Sephadex chromatography. In the presence of 0.1 mM EDTA or ethylene glycol bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), it is eluted from the column immediately before a cyclic GMP-specific phosphodiesterase, but in the presence of 0.2 mM Ca2+, the elution follows that of the cyclic GMP-specific enzyme. The two forms of the nonspecific phosphodiesterase activity are referred to as DEAD-Sephadex Fractions Ia and Ic, respectively. Their apparent molecular weights, recorded at gel filtration, vary with different preparations from 230,000 to 150,000. Occasionally, corresponding recordings for main peaks of activity also cluster round the values 120,000, 105,000, and 78,000. The enzymatic properties of Fractions Ia and Ic closely resemble each other. The enzyme activity is blocked by EDTA, partially inhibited in the presence of 1,10-phenanthroline, but only slightly affected by EGTA. The inhibitory effect of EDTA can be overcome by Mg2+ and Mn2+ and that of 1,10-phenanthroline, in part, by Zn2+; this cation in itself is inhibitory at millimolar concentrations. With submicromolar substrate concentrations, the activity of either fraction obeys linear kinetics displaying an apparent Km of approximately 0.4 micron for both substrates. Reciprocal inhibition experiments suggest that hydrolysis of both cyclic AMP and cyclic GMP is performed by the same active site. Examination of the activity using extended substrate concentration ranges indicates nonlinear kinetics; Hill plots of such data also show nonlinear curvature. The activity is inhibited by micromolar concentrations of inosine 3':5'-monophosphate (cyclic IMP), 3-isobutyl-1-methylxanthine, papervine, and some antiallergic agents. Theophylline and disodium cromoglycate are less potent inhibitors. Inhibition of activity by Lubrol PX follows a biphasic dose response curve. The activity of Fraction Ia can be enhanced 2- to 3-fold by a Ca2+-dependent activator prepared from lung tissue, whose action is counteracted by chlorpromazine, and by lysophosphatidylcholine. It is initially enhanced but subsequently decreased at exposure to trypsin. Fraction Ic is less prone to activation by these agents. The results indicate that the present activity represents an enzyme form that differs from three previously described phosphodiesterases of human lung tissue. It is apparently related to, but also shows distinct differences from the Ca2+-dependent enzyme(s) of brain and heart tissue.     

Increased activity of cyclic AMP phosphodiesterase from frozen-thawed rat liver. A role of lysosomal protease in enzyme activation.

The activity of cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in 105 000 X g supernatant fraction from frozen-thawed rat liver was 2.5 times higher than the corresponding preparation from fresh liver. This increased activity of frozen liver enzyme was accompanied by a decreased sensitivity of the enzyme to known activators such as alpha-tocopheryl phosphate and trypsin. Neither membrane-bound cyclic AMP phosphodiesterase, nor supernatant cyclic GMP phosphodiesterase increased in frozen liver preparation. It is unlikely that the activator protein of phosphodiesterase participated in the observed change of enzyme activity. Among rat tissues so far tested, the increased level of cyclic AMP phosphodiesterase was noted only in tissues rich in lysosome content. In the recombination experiment where phosphodiesterase from fresh liver was incubated with lysosomal fraction, stimulation of the enzyme activity was observed with a concomitant loss of sensitivity to above-mentioned activators. Since the stimulation by lysosomal fraction was effectively inhibited by cathepsin B1 inhibitors, leupeptin and antipain, it was deduced cathepsin-B1 (EC 3.4.12.3) type protease(s) was the main causative of activating the cyclic AMP phosphodiesterase. The freezing-thawing process of rat liver made the lysosomal membrane more permeable, and hence lysosomal proteases were released into soluble fraction during phosphodiesterase preparation. These results provide a warning not to use frozen liver for phosphodiesterase preparation, otherwise altered properties of the enzymes will be seen.     

Effects of Peptides of the Secretin-Glucagon Family and Cyclic Nucleotides on Tyrosine Hydroxylase Activity in Sympathetic Nerve Endings

Previous studies have shown that certain peptides of the secretin-glucagon family stimulate tyrosine hydroxylase activity in sympathetic neurons of the superior cervical ganglion and three of its end organs, i.e., the iris, pineal gland, and submaxillary gland. To determine whether a similar regulation occurs in other sympathetic neurons, the effects of two of these peptides, secretin and vasoactive intestinal peptide, were examined in the right cardiac ventricle of the rat, a tissue innervated primarily by the middle and inferior cervical ganglia. Both peptides stimulated tyrosine hydroxylase activity, measured in situ, in this tissue. In addition, several second messenger systems were investigated as possible mediators of this peptidergic stimulation of tyrosine hydroxylase activity in autonomic end organs. 8-Bromoadenosine 3',5'-cyclic monophosphate and forskolin elevated tyrosine hydroxylase activity in slices of both the right ventricle and the submaxillary gland. 8-Bromoguanosine 3',5'-cyclic monophosphate also stimulated tyrosine hydroxylase activity in both tissues, whereas nitroprusside stimulated activity only in the submaxillary slices. Furthermore, the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine and/or Ro 20-1724 potentiated the stimulation by secretin, as well as the stimulations by forskolin and nitroprusside. Phorbol 12,13-dibutyrate also stimulated tyrosine hydroxylase activity in cardiac and submaxillary slices; however, no potentiation of these effects was seen following addition of either phosphodiesterase inhibitor. These data, taken together with those of previous studies, suggest a role for a cyclic nucleotide, probably adenosine 3',5'-cyclic monophosphate, in the peptidergic stimulation of tyrosine hydroxylase activity in sympathetic nerve terminals.     

Calcium-dependent cyclic nucleotide phosphodiesterase. Inhibition of basal activity by heat-stable factors from rat cerebrum.

The boiled supernatant fraction from rat cerebrum contained factors which inhibited the basal activity of a Ca2+-dependent phosphodiesterase from rat cerebrum. Two inhibitory fractions were isolated by DEAE-cellulose or Sephadex chromatography and were deemed proteins, based on their sensitivity to trypsin digestion. The inhibitory fractions eluted from DEAE-cellulose columns prior to the Ca2+-dependent activator protein. The inhibitory factors, unlike the activator protein, were stable to heat treatment under alkaline conditions. The inhibitory factors caused both an increase in Km for cyclic GMP and a decrease in V. In the presence of calcium ions and purified activator protein, the Ca2+-dependent phosphodiesterase was not inhibited by the factors, but instead was slightly stimulated. The inhibitory factors caused a slight apparent stimulation of a Ca2+-independent phosphodiesterase from rat cerebrum but this proved instead to be a nonspecific stabilizing effect which was minimicked by bovine serum albumin. After prolonged alkaline treatment, the purified activator protein caused a modest Ca2+-independent activation of Ca2+-dependent phosphodiesterase. The inhibitory factors antagonized the activation of Ca2+-dependent phosphodiesterase by alkaline treated activator protein or by lysophosphatidylcholine. The inhibitory factors had no effect on activity of trypsinized Ca2+-dependent phosphodiesterase. Of various other proteins, only casein mimicked the effects of the inhibitory factors on phoshodiesterase activity.     

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.     

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.     

Activation of 3',5'-cyclic adenosine monophosphate phosphodiesterase by calcium ion and a protein activator.

3',5'-CAMP phosphodiesterase was partially purified from bovine cerebral cortex. A heat-stable activating factor was separated from the enzyme by chromatography on DEAE-cellulose. The enzyme in crude ammonium sulfate fractions was stimulated by 5 mM CaCl2. This stimulation was reversed by the calcium chelator EGTA. The main phosphodiesterase peak obtained by DEAE-cellulose chromatography was not stimulated by Ca2+. Upon addition of column effluent containing a heat stable factor, Ca2+ activation was restored. Protein activator was inactive when endogenous contaminating Ca2+ was complexed with EGTA. It was concluded that activation of phosphodiesterase requires the presence of both activator and Ca1+. From an analysis of activation of cGMP hydrolysis a kinetic model for the interaction of Ca2+ and protein activator with the phosphodiesterase was developed. Heterotropic cooperativity between the binding of Ca2+ and protein activator to the phosphodiesterase was observed, i.e., Ca1+ decreased the apparent dissociation constant for protein activator and protein activator decreased the apparent dissociation constant for Ca2+.     

Influence on adipocyte plasma membrane bound protein kinase by feedback regulator.

Protein kinase, phosphodiesterase and adenylate cyclase of plasma membrane of adipocytes and the effect of the feedback regulator (FR) on these three enzymes was measured and compared. The basal level ratio of adenylate cyclase to phosphodiesterase to protein kinase was 1:1.9:3.0. Epinephrine and/or FR alters this ratio. FR stimulated protein kinase activity up to 3 fold in the presence of a wide range of enzyme concentrations, 5-50 mug membrane protein/tube. The concentration of FR effective for stimulation of membrane protein kinase was much greater than that needed for inhibition of adenylate cyclase and phosphodiesterases. The inhibition by FR on adenylate cyclase was the most potent effect among the 3 enzymes. 1 U (or 2 U/ml) of FR inhibited 50% of the adenylate cyclase activity in a defined system. The maximum effective concentration of FR for stimulation of membrane protein kinase was greater than 10 U/ml. Histone type 11A was the best substrate for protein phosphorylation so far observed. The FR stimulatory effect was observed at all substrate concentrations used ranging from 1-5 mg/ml. A NaF concentration curve shows that 15 mM NaF gave maximum phosphorylation. The stimulatory effect of FR was observed both in the presence and absence of NaF. Protein kinase of adipocyte plasma membrane was mainly cAMP-independent. The effect of FR (20 U/ml) in stimulation of protein phosphorylation was much greater than that of cAMP (1 X 10(-6) M). The cAMP and FR effects seemed to be additive. Preincubation of plasma membrane with FR in the absence of ATP resulted in no decrease but slight increase in protein kinase activity. A shift in protein kinase, phosphodiesterase and adenylate cyclase ratios by FR suggests the regulatory role of FR in cAMP metabolism in adipocytes.     

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.     

Specificity of cGMP binding to a purified cGMP-stimulated phosphodiesterase from bovine adrenal tissue

The binding of [3H]cGMP (guanosine 3',5'-monophosphate) to purified bovine adrenal cGMP-stimulated phosphodiesterase was measured by Millipore filtration on cellulose ester filter. [3H]cGMP-binding activity was enhanced when the assay was terminated in buffer containing 70% of saturated ammonium sulfate to dilute the enzyme and wash the filters. The cGMP-binding activity was co-purified with the phosphodiesterase activity. The binding of [3H]cGMP to purified enzyme was measured in the presence or absence of the phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine. 1-Methyl-3-isobutylxanthine showed linear competitive inhibition with respect to cGMP as substrate in the phosphodiesterase reaction but stimulated the [3H]cGMP-binding activity in the binding assay. The stimulatory effect appeared not to be the result of preservation from [3H]cGMP hydrolysis; no cGMP phosphodiesterase activity has been measured under the cGMP-binding assay conditions, in the absence or presence of the inhibitor. Half-maximal stimulation by 1-methyl-3-isobutylxanthine occurred in the 5-7 microM concentration range. The specificity of binding of [3H]cGMP was investigated by adding increasing concentration of unlabeled analogs of cAMP (adenosine 3',5'-monophosphate) and cGMP. The binding of [3H]cGMP (50 nM) was displaced by unlabeled cGMP and cAMP with the following potency: 50% displacement was reached at the 0.1 microM cGMP range and only at a fiftyfold higher cAMP concentration. Our data with comparative series of analogs (e.g. 5'-amino-5'-deoxyguanosine 3',5'-monophosphate and 3'-amino-3'-deoxyguanosine 3',5'-monophosphate) showed that the potencies of stimulation of cAMP phosphodiesterase activity parallels displacement curves or [3H]cGMP binding to purified enzyme with no correlation with phosphodiesterase inhibition sequences. Those experiments suggest that the cGMP-binding activity is directly related to the non-catalytic (allosteric) cGMP-binding site.     

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

Adenosine 3',5'-cyclic monophosphate (cyclic AMP) phsophodiesterase 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.     

Cyclic nucleotide phosphodiesterases and thyroid hormones.

Evidence is presented that modulation of the maximum velocity of a particulate low K-m cyclic adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase by thyroid hormones is one mechanism for the regulation of the responsiveness of rat epididymal adipocytes to lipolytic agents such as epinephrine and glucagon. Fat cells of propylthiouracil-induced hypothyroid rats are unresponsive to lipolytic agents and the V-max of particulate low K-m cyclic AMP phosphodiesterase of these cells is elevated above normal. In vivo treatment of hypothyroid rats with triiodothyronine restores to control values both the lipolytic response of the fat cells to epinephrine and the V-max of the particulate bound low K-m cyclic AMP phosphodiesterase. No similar correlation is found with the soluble high K-m cyclic AMP phosphodiesterase. The phosphodiesterases of fat cells from normal and hypothyroid rats respond identically in vitro to propylthiouracil, triiodothyronine, methylisobutylxanthine, or theophylline, although the particulate low K-m cyclic AMP phosphodiesterase is inhibited to a greater extent than soluble cyclic guanosine 3':5'-monophosphate phosphodiesterase activity. Protein kinase of fat cells from hypothyroid rats can be stimulated by cyclic AMP to the same total activity as observed in fat cells of normal rats. However, less of the protein kinase in fat cells from hypothyroid rats was in the cyclic AMP-independent form. This shift in the equilibrium of protein kinase forms is consistent with an increased activity of low K-m cyclic AMP phosphodiesterase and probably results from a lowering of the lipolytically significant pool of cyclic AMP.     

A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.     

2-substituted derivatives of adenosine and inosine cyclic 3',5'-phosphate. Synthesis, enzymic activity, and analysis of the structural requirements of the binding locale of the 2-substituent on bovine brain protein kinase.

A number of 2-substituted cyclic nucleotide derivatives were synthesized and investigated as activators of cAMP-dependent protein kinase and as substrates for and inhibitors of cAMP phosphodiesterase. Ring closure of 5-amino-1-beta-D-ribofuranosylimidazol-4-carboxamide cyclic 3',5'-phosphate (1) with various aldehydes according to a new procedure (Meyer, R. B., Jr., Shuman, D.A., and Robins, R. K. (1974), J. Am. Chem. Soc. 96, 4962) gave new derivatives of adenosine cyclic 3',5'-phosphate with the following 2-substituents: n-propyl, n-hexl, n-octyl, n-decyl, styryl, o-methoxyphenyl, and 2-thienyl. Alkylation of 2-mercaptoadenosine cyclic 3',5'-phosphate (20, Meyer et al., 1974) gave new cAMP derivatives with the following 2-substituent: ethylthio, n-propylthio, isopropylthio, allylthio, n-decylthio, and benzylthio. Deamination of 2-methyl-,2-n-butyl-, and 2-ethylthioadenosine cyclic 3',5'-phosphate. Using multiple regression analysis, a striking relationship was found between the relative potency of the compounds as activators of bovine brain cAMP-dependent protein kinase and parameters describing the hydrophobic, steric, and electronic character of the substituents on these compounds. All compounds were substrates for a cyclic nucleotide phosphodiesterase preparation from rabbit kidney. Additionally, the compounds were as a group, good inhibitors of the hydrolysis of cAMP by phosphodiesterase preparations from rabbit lung, beef heart, and dog heart.     

Induction of phosphodiesterase by cyclic adenosine 3':5'-monophosphate in differentiating Dictyostelium discoideum amoebae.

Cyclic adenosine 3':5'-monophosphate added to the starvation media of Dictyostelium discoideum amoebae induces both intracellular and extracellular phosphodiesterase activities of these cells. The induced enzyme activity appears several hours earlier than that in starved cells which have not been induced with cyclic nucleotide. In both cases, the appearance of enzyme is inhibited by cycloheximide, and actinomycin D, and daunomycin. The KmS for the extracellular enzyme(s) of nucleotide-induced and uninduced control cells are identical. The induction of enzyme activity seems specific for cyclic adenosine 3':5'-monophosphate since cyclic guanosine 3':5'-monophosphate, as well as other nucleotides, have no effect. No differences in the activity or excretion of either N-acetylglucosaminidase or the inhibitory of the extracellular phosphodiesterase are observed between cyclic adenosine 3':5'-monophosphate-induced and control cells. A direct activation of phosphodiesterase by cyclic adenosine 3':5'-monophosphate can be excluded, since the addition of this nucleotide to cell lysates has no effect on the enzyme activity.     

Regulation of cyclic nucleotide phosphodiesterase activity in human lung fibroblasts.

Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.     

Protein inhibitor of cyclic adenosine 3':5'-monophosphate phosphodiesterase in retina.

A protein acting as inhibitor of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.1.) activity was found in the ox retina tissue. An inhibitor from one tissue (ox retina) effectively cross-inhibited a phosphodiesterase from another tissue (rat brain), indicating a lack of tissue specificity. Kinetic analysis showed that inhibition was independent of the time of preliminary incubation of the inhibitor with enzyme but dependent on its concentration in the reaction mixture. An inhibitor decreased the V of the enzyme and had no effect on its Km for cyclic adenosine-3':5'-monophosphate. The inhibitory effect was more pronounced with cyclic adenosine-3':5'-monophosphate than with cyclic guanosine-3':5'-monophosphate used as substrates of the reaction. The extractable form of the phosphodiesterase of the retina rod outer segments was much more sensitive to the inhibitory action than the membrane-bound one. The binding of labeled cyclic adenosine-3':5'-monophosphate to the inhibitory protein was shown not to occur. The inhibitor was sensitive to trypsin treatment, indicating that it was a proten attempt was mode to purify the inhibitory factor. Gel filtration indicated that the inhibitor had a molecular weight of 38 000.