Phospholipid modulation of low-Km cyclic AMP phosphodiesterase activity on rat adipocyte microsomes

The ability of nine phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase was examined in microsomal fractions of rat adipocytes. The enzyme was activated by phosphatidylserine (21% at 300 microM) and phosphatidylglycerol (36% at 300 microM). The activation was concentration dependent over the range 1-1000 microM. Six other phospholipids were without effect. Phosphatidylinositol 4-phosphate inhibited the activity of the enzyme over the same range of concentrations (26% at 300 microM). Phosphatidylserine also activated a partially purified preparation of the enzyme, whereas phosphatidylinositol 4-phosphate was ineffective. The mechanism of the activation of the enzyme by phosphatidylserine and phosphatidylglycerol involved an increase in the apparent Vmax of the enzyme, while the inhibition by phosphatidylinositol 4-phosphate was associated with an increase in the Km of the enzyme for substrate. The phospholipid modulators of low-Km cyclic AMP phosphodiesterase activity did not alter the activity of high-Km cyclic AMP phosphodiesterase. The ability of phospholipids to alter the activity of low-Km cyclic AMP phosphodiesterase in native membranes suggests a possible role for phospholipids in metabolic regulation.     

Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart.

1. Isoelectric focusing on a flat gel bed of the rat heart cytosolic fraction resolved cyclic nucleotide phosphodiesterase activity into several forms, characterized by their substrate specificity, kinetic constants and dependence towards Ca2+ and calmodulin. A peak of pI 4.9 displayed 20 times more affinity for cyclic GMP than for cyclic AMP and was markedly inhibited by EGTA. A less substrate-specific form, only slightly sensitive to EGTA inhibition, focused at pH 5.45. Several overlapping peaks detected between pH 5.55 and pH6 specifically hydrolysed cyclic AMP, with non-Michaelian kinetics; these peaks were insensitive to Ca2+ chelation. 2. Isoelectric focusing did not dissociate enzyme-calmodulin complexes, as none of the resulting peaks was activatable by calmodulin plus Ca2+. 3. Some new information on rat cardiac phosphodiesterase is obtained with this technique, which is convenient for routine analytical studies of phosphodiesterase, as well as for preparative purposes.     

Dibutyryl cyclic AMP increases phosphodiesterase activity in the rat heart

The influence of increasing the in vivo concentration of cyclic AMP on the activity of cyclic nucleotide phosphodiesterase (PDE) in rat heart was investigated. One, three, and five hourly injections of 5.0 mg dibutyryl (Bt2) cyclic AMP significantly increased the activity of PDE in the supernatant fraction of rat heart using 1.0 microM cyclic AMP as the assay substrate concentration. When 100 microM cyclic AMP was used in the assay reaction, increases in enzymes activity were seen following five and eight nucleotide injections. The nucleotide-induced increase in PDE activity was dose dependent. When the five-injection protocol was used, PDE activity remained elevated for at least 4 h, while activity had returned to control levels within this time when two hourly injections were used. The nucleotide stimulation of PDE activity was blocked by cycloheximide. Five hourly infections of Bt2 cyclic AMP increased PDE activity in the liver and fast-twitch red muscle. A reduction in PDE activity in fast-twitch white muscle was seen following nucleotide injections. These findings are consistent with the hypothesis that prolonged elevations in the intracellular concentration of cyclic AMP cause an elevation in myocardial PDE activity. The increased activity seems to be the result of protein synthesis. These data suggest that cyclic AMP contributes significantly in regulating its own metabolism in the rat heart.     

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.     

Purification of rat heart cyclic nucleotide phosphodiesterase on column of immobilized phenylbutenolide inhibitor

Cyclic nucleotide phosphodiesterase was purified over 200-fold in a single step from the rat heart cytosolic fraction, using affinity chromatography on phenylbutenolide inhibitor immobilized to AH Sepharose. After elimination of the contaminating proteins by washing with the loading buffer and then with 0.4 M KCl buffer, without any loss in enzymatic activity, the cyclic nucleotide phosphodiesterase was eluted in good zields with a linear KCl gradient from 0.4 M to 1.8 M. Enzymatic activity determination performed with both cyclic AMP and cyclic GMP as substrate, either at low (0.25 μM) or at high (25 μM) concentration, pointed out the presence of several phosphodiesterase forms with different substrate specificities, in the elution profiles.     

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

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

Characterization of cyclic nucleotide phosphodiesterase activity in Phaseolus vulgaris

Cyclic nucleotide phosphodiesterase (3',5'-cyclic nucleotide nucleotidohydrolase, EC 3.1.4.17) activity isolated from Phaseolus vulgaris L. cv. Limberg seedlings was partially purified and characterized by fractional (NH₄)₂SO₄ precipitation, DEAE-cellulose chromatography, chromatography on 3',5'-cAMP-agarose, gel permeation chromatography and chromatofocusing. A crude enzyme preparation, a 30–65% (NH₄)₂SO₄ pellet, showed an acidic pH optimum. The enzyme activity was stimulated by imidazole and divalent cations such as Ca²⁺, Mg²⁺ and Mn²⁺, whereas NaF, PP_i and Fe³⁺ were inhibitory. Isobutylmethylxanthine had no significant effect on the plant enzyme. An M_I of 42 000 was estimated by gel permeation high performance liquid chromatography. By chromatography on 3',5'-cAMP-agarose a phosphodiesterase was resolved that produced 5'-AMP as sole reaction product.     

Heat-stable calmodulin-binding protein in rat testis. Inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase activity

An inhibitor of procine brain calmodulin-dependent cyclic nucleotide phosphodiesterase was purified about 940-fold from rat testis. This inhibitor inhibited the calmodulin-induced activation of the enzyme without affecting its basal activity. The inhibitor activity was counteracted by a high concentration of calmodulin, but was not by a high concentration of Ca2+. The analysis on polyacrylamide disc gel electrophoresis demonstrated that the inhibitor and calmodulin form a complex in the presence of Ca2+ but not in the presence of excess amount of EGTA. This inhibitor also inhibited the calmodulin-induced activation of Ca2+, Mg2+ -ATPase of human erythrocytes. The inhibitor appeared to be a heat-stable protein, since the inhibitor activity was not attenuated by boiling up to 9 min but was completely abolished by tryptic or chymotryptic digestion. The molecular weights of the inhibitor determined by linear polyacrylamide gradient gel electrophoresis under nondenaturing conditions and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 40,000 and 32,000, respectively. Thus, the inhibitor is suggested to be a calmodulin-binding protein composed of a monomer which has unique properties different from those of other tissues.     

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 K_m for cyclic GMP and a decrease in $\text{V}$. 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 mimicked 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 phosphodiesterase activity.     

Detection of a calmodulin-sensitive cyclic nucleotide phosphodiesterase in rat parotid gland

Calmodulin coupled to Sepharose has provided a rapid and sensitive means of isolating a cyclic nucleotide phosphodiesterase activity which is stimulated by the calmodulin-Ca²⁺ complex, from rat parotid gland. Initial experiments established that phosphodiesterase activity sensitive to calmodulin and Ca²⁺ could not be demonstrated in crude extracts of rat parotid gland or after partial purification of rat parotid phosphodiesterase over DEAE-cellulose. However, it was possible to readily demonstrate the presence of a cyclic nucleotide phosphodiesterase activity regulated by calmodulin if the extracts were first purified by batch ion-exchange chromatography over DEAE-cellulose followed by affinity chromatography with calmodulin coupled to Sepharose. The batch ion-exchange chromatography step removed the major portion of free parotid calmodulin which could compete with calmodulin-coupled Sepharose for the proteins regulated by calmodulin. Thus, by employing an initial chromatography step over DEAE-cellulose to separate phosphodiesterase activity from calmodulin, it was possible to increase the recovery of calmodulin-sensitive phosphodiesterase after affinity chromatography with calmodulin coupled to Sepharose. This approach should be useful for demonstrating the presence of and for purifying other parotid proteins regulated by calmodulin.     

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.     

Determination of cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography

A new assay for cyclic nucleotide phosphodiesterase activity by high-performance liquid chromatography with on-line radiochemical detection has been developed. The method is based on the measurement of 3H-labeled nucleoside monophosphates formed from cyclic nucleotides by the action of 3',5'-cyclic-nucleotide phosphodiesterase (PDE). The reaction products are determined from the incubation mixture after removal of the protein by injection of an aliquot into the liquid chromatograph. The detection limit with counting efficiency of 30% is 20 fmol of 3H-labeled product, which makes the method suitable for detection of low PDE activities.     

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

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

Cyclic nucleotide phosphodiesterase activity in rat adrenal gland zones

The distribution of cyclic 3′, 5′ -nucleotide phosphodiesterase activity in the rat adrenal gland has been studied. Phosphodiesterase activity was 10-fold higher in the zona glomerulosa than in the zona fasciculata-reticularis. Kinetic studies carried out at low substrate concentrations suggest the possible presence of multiple forms of phosphodiesterase activity in both zones of the adrenal; however, these forms appear to have similar apparent Km's for cAMP. Thus, the well known differences in the steroidogenic response of the two zones to ACTH stimulation may be partially explained by large differences in total activities of the various forms of phosphodiesterase.     

Amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase from bovine heart

The complete amino acid sequence of the cyclic GMP stimulated cyclic nucleotide phosphodiesterase (cGS-PDE) of bovine heart has been determined by analysis of five digests of the protein; placement of the C-terminal 330 residues has been confirmed by interpretation of the corresponding partial cDNA clone. The holoenzyme is a homodimer of two identical N alpha-acetylated polypeptide chains of 921 residues, each with a calculated molecular weight of 103,244. The C-terminal region, residues 613-871, of the cGS-PDE comprises a catalytic domain that is conserved in all phosphodiesterase sequences except those of PDE 1 from Saccharomyces cerevisiae and a secreted PDE from Dictyostelium. A second conserved region, residues 209-567, is homologous to corresponding regions of the alpha and alpha' subunits of the photoreceptor phosphodiesterases. This conserved domain specifically binds cGMP and is involved in the allosteric regulation of the cGS-PDE. This regulatory domain contains two tandem, internal repeats, suggesting that it evolved from an ancestral gene duplication. Common cyclic nucleotide binding properties and a distant structural relationship provide evidence that the catalytic and regulatory domains within the cGS- and photoreceptor PDEs are also related by an ancient internal gene duplication.     

Age-related alterations in cyclic nucleotide phosphodiesterase activity in dystrophic mouse leg muscle

Previous reports have described both increased and decreased cyclic nucleotide phosphodiesterase (PDE) activity in dystrophic muscle. Total PDE activity was measured in hind leg muscle from a mouse model of Duchenne muscular dystrophy (mdx) and a genetic control strain at 5, 8, 10, and 15 weeks of age. Total PDE activity declined in fractions isolated from mdx muscle over this time period, but was stable in fractions from control mice. Compared with age-matched controls, younger mdx muscle had higher cAMP and cGMP PDE activity. However, at 15 weeks, fractions from both strains had similar cGMP PDE activity and mdx fractions had lower cAMP PDE activity than controls. Particulate fractions from mdx muscle showed an age-related decline in sensitivity to the PDE4 inhibitor RO 20-1724. A similar loss of sensitivity to the PDE2 inhibitor erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA) was seen in a particulate fraction from mdx muscle and to a lesser degree in control muscle. These results suggest that the earlier disagreement regarding altered cyclic nucleotide metabolism in dystrophic muscle may be due to changes with age in PDE activity of dystrophic tissue. The age-related decline in particulate PDE activity seen in dystrophic muscle appears to be isozyme-specific and not due to a generalized decrease in total PDE activity.