CYCLIC NUCLEOTIDE METABOLISM IN ELECTROPLAX OF ELECTROPHORUS ELECTRICUS

Abstract— In order to describe the regulation of cyclic nucleotide metabolism in a cholinergic tissue, the properties of cyclic nucleotide phosphodiesterase were determined in electroplax of Electrophorus electricus and compared to those of mammalian brain. Electroplax phosphodiesterase was Mg²⁺ -dependent. localized in the soluble fraction and displayed normal linear Lineweaver-Burk kinetics ( K _m: cyclic AMP. 1.4 μ m ; cyclic GMP, 0.54 μ m ). No low affinity (i.e. high K _m) activity was detected. These results were correlated with comparatively low tissue levels of cyclic AMP (67 pmol/g) and cyclic GMP (3.2 pmol/g). Attempts were made to detect calcium-dependent phosphodiesterase because of the presence of large amounts of the calcium-dependent regulator protein (CDR) in electroplax, as this protein has been shown to activate brain phosphodiesterase. Assay with EGTA under a variety of conditions revealed that no calcium-dependent activity could be detected. Preparation of CDR-deficient phosphodiesterase also failed to produce calcium-dependent activity. Assay of phosphodiesterase in other cholinergic tissues revealed calcium-dependent activity in Electrophorus muscle and rat diaphragm but not in Torpedo electroplax. The results suggest that calcium-dependent activity is not a significant portion of phosphodiesterase in electroplax and indicate alternate roles for CDR in electric tissue.     

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.     

Cross-linking of iodine-125-labeled, calcium-dependent regulatory protein to the Ca2+-sensitive phosphodiesterase purified from bovine heart.

The calcium-dependent regulatory protein (CDR).Ca2+ sensitive cyclic nucleotide phosphodiesterase was purified to apparent homogeneity from bovine heart by using ammonium sulfate fractionation, DEAE-ceelulose chromatography, and CDR-Sepharose affinity chromatography. The enzyme was purifed 13 750-fold with a 10% yield and a specific activity of 275 mumol of cAMP min-1 mg-1. The purified enzyme ran as a single band during sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 57 000. Phosphodiesterase activity was stimulated 10-fold by Ca2+ and CDR with half-maximal activation occurring at 9 ng/assay. [125I]CDR was cross-linked to the purified phosphodiesterase by using dimethyl suberimidate Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cross-linked products revealed a number of discrete 125I-labeled bands. The molecular weights of the cross-linked products indicate that the stoichiometry of the phosphodiesterase complex is A2C2, where A is the phosphodiesterase catalytic subunit and C is the calcium-dependent regulatory protein.     

Calcium-dependent cyclic nucleotide phosphodiesterase from glial tumor cells

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been identified in homogenates of C-6 glial tumor cells. The Ca²⁺-dependent phosphodiesterase was resolved by ECTEOLA-cellulose chromatography into two fractions. One fraction contained a protein regulator of the enzyme which was identical to a homogeneous Ca²⁺-binding protein (CDR) from porcine brain by the criteria of electrophoretic migration, biological activity, heat stability, and behavior in diverse chromatographic systems. The second fraction contained deactivated enzyme (CDR-dependent phosphodiesterase) which regained full activity upon the readdition of both Ca²⁺ and CDR. In subcellular fractionation experiments both the CDR and the Ca²⁺-dependent phosphodiesterase were predominantly located in the 100,000g supernatant fraction.The apparent K_m values of the phosphodiesterase for cyclic AMP (cAMP) and cyclic GMP (cGMP) were 10 and 1.2 μm, respectively, when CDR was not rate limiting. Minor increases in the apparent K_m for cAMP were observed at rate-limiting concentrations of CDR. At the ratio of CDR to CDR-dependent enzyme present in the C-6 cell homogenate, half-maximal activation was conferred by 4 μm Ca²⁺ for the hydrolysis of 25 μm cGMP and by 8 μm Ca²⁺ for the hydrolysis of 25 μm cAMP. Increased ratios of CDR to CDR-dependent phosphodiesterase increased the sensitivity of the enzyme to Ca²⁺. The enzyme was more sensitive to CDR with cGMP as substrate than with cAMP, and more sensitive at high than at low cyclic nucleotide substrate concentrations. The quantity of enzyme in the assay also influenced the amount of CDR required for half-maximal activation.     

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.     

Detection of calcium-dependent regulatory protein binding components using 125I-labeled calcium-dependent regulatory protein.

The calcium-dependent regulatory protein (CDR) purified from bovine brain was iodinated with Na[125I]I using the lactoperoxidase-glucose oxidase system. The iodinated protein retained its ability to stimulate the Ca2+-sensitive CDR-depleted cyclic nucleotide phosphodiesterase from bovine heart. Stimulation of the phosphodiesterase by 125I-CDR was Ca2+-dependent and the labeled protein had a Ka for activation of cyclic nucleotide phosphodiesterase that was 4 times greater than unmodified CDR. 125I-CDR formed a Ca2+-dependent complex with the partially purified cyclic nucleotide phosphodiesterase which was detectable by autorradiography following electrophoresis of the complex on nondenaturing gels. This technique was used to detect CDR binding components in crude homogenates prepared from bovine heart and brain.     

Lack of altered cyclic nucleotide phosphodiesterase activity in the aorta and heart of the spontaneously hypertensive rat

DEAE-cellulose chromatography demonstrated that the levels of the individual cyclic nucleotide phosphodiesterase were unchanged in the aorta and heart of the spontaneously hypertensive rat as compared with the normotensive control rat. Three peaks of cyclic nucleotide phosphodiesterase activity were observed in both heart and aorta. Peak I enzyme hydrolyzed predominantly cyclic GMP while peak III enzyme hydrolyzed predominantly cyclic AMP. Peak II enzyme was less specific but hydrolyzed more cyclic GMP than cyclic AMP The levels of phosphodiesterase activator in aorta and the responsiveness of peaks I and II from aorta and heart to activator were unchanged in the hypertensive rat. Therefore the decrease in cyclic AMP levels observed by others in aorta and heart of the spontaneously hypertensive rat were probably not due to altered phosphodiesterase activity.     

Effects of spermine on activity and stability of calcium-dependent guanosine 3',5'-monophosphate phosphodiesterase.

Spermine in micromolar concentrations decreased the basal activity of a guanosine 3',5'-monophosphate (cGMP) phosphodiesterase from bovine brain but had no effect in the presence of Ca2+ plus the calcium-dependent regulatory protein (CDR) which increased the activity of the enzyme 4- to 6-fold. Similar effects of spermine were observed on the enzyme at several stages of purification. Spermidine and putrescine were also inhibitory but higher concentrations were required. In the absence of Ca2+ and CDR, the enzyme exhibited two apparent Km values for cGMP (2.5 and 20 microM) which were unaltered by spermine. In the presence of Ca2+ and CDR (when spermine had no effect on activity), a single Km (3.5 microM) was observed. Enzyme purified by chromatography on CDR-Sepharose was rapidly inactivated during incubation at 30 degrees C in 5 mM potassium phosphate buffer (pH 7.0) with EDTA and ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA). Spermine (20 microM) partially stabilized enzyme activity under these conditions, although it was somewhat less effective than 2 mM MgCl2. The inhibitory effects of spermine (or other polyamines) on basal phosphodiesterase activity, which can be overcome by Ca2+ and CDR, could be important in the regulation of cellular cyclic nucleotide content.     

Calmodulin in mouse male germ cells: a qualitative and quantitative study

Isolated male germ cells of the mouse possess a heat-stable stimulatory activity of Ca2+-dependent, calmodulin-free phosphodiesterase. Ionic exchange chromatography allowed partial purification of the activator and the isolation of multiple forms of phosphodiesterase stimulation inhibitor. The activator has been identified as calmodulin on the basis of chromatographic behaviour and electrophoretic mobility. Quantitative analysis showed variations of calmodulin levels at different stages of spermatogenesis. Quantitative analysis of cyclic nucleotide hydrolysis in germ cell cytosol showed that the activity of Ca2+-dependent phosphodiesterase is different in meiotic and post-meiotic mouse male germ cells. These data suggest that calcium-dependent pathway and a Ca2+-dependent regulation of cyclic nucleotides are present in developing germ cells.     

Regulation of the high Km cyclic nucleotide phosphodiesterase of adrenal medulla by the endogenous calcium-dependent-protein activator.

A cyclic nucleotide phosphodiesterase sensitive to the calcium-dependent endogenous protein activator has been identified in rat and beef adrenal medulla. In this tissue the ratio between the activity of this enzyme and that of the low Km enzyme is smaller than the corresponding ratio in rat brain. The activator-sensitive phosphodiesterase, isolated from beef adrenal medulla has a high Km for cyclic 3,5-AMP. Saturating concentrations of the calcium dependent protein activator decreased significantly the apparent Km of this enzyme using cAMP as a substrate.     

Adrenal medullary cyclic nucleotide phosphodiesterase: lack of activation by the calcium-dependent regulator.

Calcium-dependent regulator, a calcium-binding protein isolated from brain and adrenal medulla, has been shown to activate a brain calcium-sensitive cyclic nucleotide phosphodiesterase. To determine if this protein has the same role in the adrenal medulla, the cyclic nucleotide phosphodiesterase of adrenal medulla was characterized. Neither crude nor partially purified adrenal medullary phosphodiesterase was inhibited by EGTA or stimulated by calcium and the calcium-dependent regulator, whereas similar brain preparations displayed sensitivity to these agents. As the calcium-dependent regulator does not appear to stimulate adrenal medullary cyclic nucleotide phosphodiesterase activity, alternate roles of this protein in adrenal medulla are suggested.     

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.     

Properties of cyclic nucleotide phosphodiesterase in the central nervous system of Manduca sexta.

The properties of cyclic nucleotide phosphodiesterase were studied in soluble and particulate fractions from the central nervous system of Manduca sexta (Lepidoptera: Sphingidae). It was determined that: (1) The highest levels of phosphodiesterase occur in nervous tissue. (2) The total and specific enzyme activities of larval and adult brains are greater than those of the remaining ganglia. (3) Specific central nervous sy stem phosphodiesterase activities of the adult are lower than those of the larva, but both protein and total phosphodiesterase contents are considerably greater in the adult central nervous system. (4) Mg2+ is not absolutely required for either cyclic AMP-phosphodiesterase or cyclic GMP-phosphodiesterase activity. (5) Phosphodiesterase is inhibited by a variety of physiological and non-physiological compounds, nucleoside triphosphates being particularly effective; Some potent inhibitors of mammalian phosphodiesterase are comparatively ineffective toward Manduca sexta phosphodiesterase. (6) Kinetic analyses of soluble and particulate phosphodiesterase revealed non-linear double-reciprocal plots for the hydrolysis of both cyclic AMP and cyclic GMP, with Michaelis constants of approximately 10 mu M and 20 mu M; (7) The hydrolysis of both cyclic nucleotides appears in part to be the function of a single enzyme or related enzymes in the insect central nervous system. It follows that the intracellular level of one cyclic nucleotide may influence the concentration of the other by inhibiting its DEGRADATION.     

Calcium-dependent interactions of an ionophore A23187 with calmodulin

We found that ionophore A23187 interacted reversibly with calmodulin (CaM), in a calcium-dependent fashion. It was found that A23187 interacts selectively with CaM, among calcium binding proteins (such as troponin C and S-100 protein) and other proteins. However, apparently differing from W-7, A23187 did not suppress CaM-dependent enzyme activity such as myosin light chain kinase and Ca2+-dependent cyclic nucleotide phosphodiesterase. Our observations suggest that there are novel calcium-dependent regions of CaM which can be monitored using ionophore A23187 and may not be related to enzyme activation.     

Assessment of selective inhibition of rat cerebral cortical calcium-independent and calcium-dependent phosphodiesterases in crude extracts using deoxycyclic AMP and potassium ions

The effects of various inhibitors on the activity of calcium-independent and calcium-dependent phosphodiesterases from rat cerebral cortex were examined. While the agents varied greatly in their relative potency, each was found to be approximately equipotent in inhibiting the calcium-dependent hydrolysis of either cyclic AMP or cyclic GMP. In contrast, the inhibitors displayed a marked substrate specificity for the calcium-independent enzyme with ratios of IC₅₀ values for inhibition of cyclic GMP hydrolysis when compared to cyclic AMP hydrolysis in decreasing order being: ZK 62711 (? 100) > Ro 20–1724 (?>25) papaverine (13) > 7-benzyl IBMX (4) > quercetin and kaempferol (2). The differential selectivity of the inhibitors for the two enzymes was most pronounced for ZK 62711 and Ro 20–1724 which were at least 25–100-times more potent in inhibiting the calcium-independent hydrolysis of cyclic AMP when compared to the calcium-dependent hydrolysis of cyclic AMP. In contrast, 7-benzyl IBMX, kaempferol and quercetin were 8–100-times more effective as inhibitors of cycluc GMP hydrolysis by the calcium-dependent phosphodiesterase while 7-benzyl IBMX and trimazosin displayed a similar enzyme selectivity using cyclic AMP as substrate. With the exception of papaverine, all agents were competitive inhibitors of the calcium-dependent phosphodiesterase. The type of inhibition observed with the calcium-independent enzyme was dependent on the substrate employed. The specificity of potassium ions in inhibiting the activity of the calcium-dependent phosphodiesterase and deoxycyclic AMP in inhibiting the calcium-independent enzyme was found to provide a convenient means to assess the effects of agents on these activities in crude extracts of cerebral cortex.     

Cyclic nucleotide phosphodiesterase in rat neostriatum: multiple isoelectric forms with similar kinetic properties

Separation of multiple forms of cyclic nucleotide phosphodiesterase from the soluble supernatant fraction of rat neostriatum by isoelectric focusing yielded five separate peaks of cyclic nucleotide hydrolysing activity. Each separated enzyme form displayed a complex kinetic pattern for the hydrolysis of both cyclic AMP and cyclic GMP, and there were two apparent Km's for each nucleotide. At 1 microM substrate concentration, four enzyme forms exhibited higher activity with cyclic AMP than with cyclic GMP, while one form yielded higher activity with cyclic GMP than with cyclic AMP. Cyclic AMP and cyclic GMP were both capable of almost complete inhibition of the hydrolysis of the other nucleotide in all the peaks separated by isoelectric focusing; the IC50's for this interaction correlated well with the relative rates of hydrolysis of each nucleotide in each peak. The ratio of activity at 1 microM substrate concentration for the five enzyme forms separated by isoelectric focusing was 10:10:5:15:1 for cyclic AMP hydrolysis; and 6:6:4:8:2 for cyclic GMP hydrolysis; and the isoelectric points of the five peaks were 4.3, 4.45, 4.7, 4.85, and 5.5, respectively. Known phosphodiesterase inhibitors did not preferentially inhibit any of the separated forms of activity for either cyclic AMP or cyclic GMP hydrolysis, at either high (100 microM) or low (1 microM) substrate concentrations. Preliminary examination of the subcellular distribution of the different forms of enzyme activity indicated a different degree of attachment of the various forms to particulate tissue components. Isoelectric focusing of the soluble supernatant of rat cerebellum gave rise to a slightly different pattern of isoelectric forms from the neostriatum, indicating a different cellular distribution of the isoelectric forms of PDE in rat brain. Polyacrylamide disc gel electrophoresis of the soluble supernatant of rat neostriatum also generated a characteristic pattern of five separate peaks of cyclic nucleotide phosphodiesterase activity, each of which hydrolysed both cyclic AMP and cyclic GMP. Polyacrylamide gel electrophoresis of single enzyme forms previously separated by isoelectric focusing gave single peaks, with a marked correspondence between the enzyme forms produced by isoelectric focusing and those produced by gel electrophoresis, suggesting that both protein separation procedures were isolating the same enzyme forms. The results indicate the existence of multiple isoelectric forms of cyclic nucleotide phosphodiesterase in the soluble supernatant fraction of rat neostriatum, all of which exhibit similar properties. In this tissue a single kinetic form of this enzyme appears to exist displaying complex kinetic behaviour indicative of negative cooperativity and hydrolysing both cyclic AMP and cyclic GMP, with varying affinities.     

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.     

Calcium-dependent 3':5'-cyclic nucleotide phosphodiesterase. Inhibition of basal activity at physiological levels of potassium ions.

A calcium-dependent cyclic nucleotide phosphodiesterase from rat cerebrum was, in the absence of activator protein, inhibited by various monovalent cations. The inhibition was rapid, readily reversible, and concentration-dependent, with 100 mM cesium, rubidium, or potassium ion inhibiting essentially all basal enzyme activity, while 100 mM sodium or lithium ions produced only moderate inhibition. The potency of the cations in inhibiting the enzyme was Cs greater than or equal to Rb greater than K greater than Na greater than or equal to Li. Potassium ions increased the apparent Km for cyclic GMP and cyclic AMP by 3- and 5-fold, respectively. At 100 mM, the monovalent cations inhibited enzyme activated by the calcium-dependent activator by only 15 to 30%, while at 55 mM no inhibition pertained. Potassium and sodium ions at 55 mM had no effect on the calcium-independent phosphodiesterase from rat cerebrum. The results indicate that at normal intracellular concentrations of potassium ions the activity of the calcium-dependent phosphodiesterase is virtually completely dependent on the presence of calcium plus activator protein.     

Cyclic nucleotide phosphodiesterases in the cricket, Acheta domesticus.

Exceptionally high levels of guanosine 3'-5'-cyclic monophosphate (cyclic GMP) in the accessory reproductive gland of the male house cricket, Acheta domesticus, led to an investigation of cyclic nucleotide phosphodiesterase (EC 3.1.4.--) as a possible regulatory enzyme. Cricket cyclic nucleotide phosphodiesterase activity with cyclic GMP or cyclic AMP as substrate had a pH optimum around 9.0, required Mg2+ or Mn2+ for maximal activity, and was inhibited by EDTA and methylxanthines. Cyclic GMP phosphodiesterase occurred mainly in the soluble fraction of homogenates of accessory glands or whole crickets, but cyclic AMP phosphodiesterase in the accessory gland was primarily particulate. Kinetic analysis indicated three forms of cyclic GMP phosphodiesterase, with Km values at 2.9 muM, 71 muM and 1.5 mM. Chromatography of whole cricket or accessory gland extracts on DEAE cellulose gave an initial peak having comparable activity with either cyclic GMP or cyclic AMP, and a second peak specific for cyclic AMP. There were no appreciable changes in the specific activity or kinetic properties of accessory gland cyclic GMP phosphodiesterase during a developmental period over which cyclic GMP levels rise more than 500-fold. Thus, the accumulation of cyclic GMP in the accessory gland is probably not associated with concomitant developmental modulation of phosphodiesterase activity.     

Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase activities of rat mammary tissue.

Cyclic nucleotide phosphodiesterase activity in mammary tissue from rats in midlactation was resolved by DEAE-cellulose chromatography into three functionally distinct fractions: a Ca2+/calmodulin-stimulated cyclic GMP phosphodiesterase, a cyclic GMP-stimulated low-affinity cyclic nucleotide phosphodiesterase, and a high-affinity cyclic AMP-specific phosphodiesterase. The absolute activities and relative proportions of high- and low-affinity enzymes resemble those found, for example, in liver, as distinct from those in excitable tissues. Three functional characteristics are described which are peculiar to mammary-tissue phosphodiesterases. Firstly, the concentration of free Ca2+ required to achieve half-maximal activation of the Ca2+/calmodulin-stimulated phosphodiesterase is somewhat higher than for the analogous enzyme in other tissues; secondly, the activity of this enzyme towards cyclic AMP relative to that towards cyclic GMP is unusually low, and thirdly, the low-affinity cyclic nucleotide phosphodiesterase is inhibited by low concentrations of free Ca2+.