Ca2+-dependent cyclic nucleotide phosphodiesterase from rabbit lung.

Studies are presented which demonstrate that rabbit lung contains both Ca²⁺-activated cyclic nucleotide phosphodiesterase and calmodulin activity. The Ca²⁺-activatable cyclic nucleotide phosphodiesterase is different from the common type in that it contains tightly bound calmodulin. The bound calmodulin is not dissociated from the enzyme even in very low concentrations of Ca²⁺ after DEAE-cellulose and Sephadex G-200 column chromatography.     

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)     

Sequence homology of the Ca2+-dependent regulator of cyclic nucleotide phosphodiesterase from rat testis with other Ca2+-binding proteins.

A Ca2+-dependent regulator protein of cyclic 3':5'-nucleotide phosphodiesterase (EC 3.1.4.17) has previously been isolated from rat testis and shown to be a heat-stable, Ca2+-binding protein with a molecular weight of approximately 17,000. The Ca2+-dependent regulator protein is also structurally similar to troponin-C, the Ca2+-binding component of muscle troponin and Ca2+ mediator of muscle contraction. The present report describes a partial amino acid sequence of the Ca2+-dependent regulator. The protein (148 amino acids) is 50% homologous with skeletal muscle troponin-C, but is 11 residues shorter than the muscle protein. The Ca2+-dependent regulator protein has an NH2-terminal sequence of acetyl-Ala-Asp-Glu, a COOH-terminal sequence of Thr-Ala-Lys and 1 residue of epsilon-trimethyllysine located at position 115. All of these properties are distinct from those of other homologous Ca2+-binding proteins. These properties may account for the biological specificities demonstrated by these proteins as compared to the Ca2+-dependent regulator protein. Based on the sequence and a comparison of the Ca2+-dependent regulator protein to other calcium-binding proteins, our data support the view that all of these moecules contain common sequences, especially at their proposed metal-binding sites.     

Calcium-independent bacterial activator of cyclic nucleotide phosphodiesterase and Ca2+/Mg2+-ATPase

Ca2+-independent protein-modulator (BacM) was found in the culture medium of Staphylococcus aureus. BacM activated calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+/Mg2+-ATPase in the same way as calmodulin. BacM was shown to be a proteolytic fragment of the exotoxin secreted by the S. aureus strain under study. The kinetic analyses of the ATPase activation by BacM and CaM were performed. These studies demonstrated that the enzyme molecule contains at least two activator-sensitive sites. Experiments on the ATPase activation by Ca2+ both in the presence and in the absence of BacM and CaM documented that CaM-ATPase and BacM-ATPase complexes can exist at low concentrations of calcium. Analysis of activation curves of ATPase by Ca2+ revealed three Ca2+-binding sites in the enzyme-activator complex.     

Ca2+-dependent cyclic nucleotide phosphodiesterase is activated by poly(L-aspartic acid)

Ca2+-dependent cyclic nucleotide phosphodiesterase (Ca2+-PDE) activity was stimulated by poly(L-aspartic acid) but not by poly(L-glutamic acid), poly(L-arginine), poly(L-lysine), and poly(L-proline). This activation was Ca2+ independent and did not further enhance the activation of Ca2+-PDE by Ca2+-calmodulin (CaM). Poly(L-aspartic acid) produced an increase in the Vmax of the phosphodiesterase, associated with a decrease in the apparent Km for the substrate, such being similar to results obtained with Ca2+-CaM. Poly(L-aspartic acid) did not significantly stimulate myosin light chain kinase and other types of cyclic nucleotide phosphodiesterase. CaM antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), trifluoperazine, and chlorpromazine selectively antagonized activation of the enzyme by poly(L-aspartic acid). Kinetic analysis of W-7-induced inhibition of activation of phosphodiesterase by poly(L-aspartic acid) was in a competitive fashion, and the Ki value was 0.19 mM. On the other hand, prenylamine, another type of calmodulin antagonist that binds to CaM at sites different from the W-7 binding sites, did not inhibit the poly(L-aspartic acid)-induced activation of Ca2+-dependent cyclic nucleotide phosphodiesterase. These results imply that poly(L-aspartic acid) is a calcium-independent activator of Ca2+-dependent phosphodiesterase and that aspartic acids in the CaM molecule may play an important role in the activation of Ca2+-PDE.     

Purification of a Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart by specific interaction with its Ca2+-dependent modulator protein.

A Ca2+-activatable cyclic nucleotide phosphodiesterase from bovine heart can be eluted from a DEAE-cellulose column either in the free form by buffers containing 0.1 mM ethylene glycol bis(beta-aminoethyl ether)N-N,N'N'-tetraacetic acid (EGTA) or as a complex of the enzyme with its protein modulator by buffers containing 0.01 mM CaCl2. A purification procedure based primarily on the significantly different affinity of the two forms of the enzyme for DEAE-cellulose was developed for the purification of the enzyme from bovine heart. The procedure involves ammonium sulfate fractionation, three chromatographic steps on DEAE-cellulose, and gel filtration on Sephadex G-200 with a 5000-fold purification over the crude extract. The purified enzyme has a specific activity of 120 mumol of cAMP/mg/min, can be activated 5-fold by Ca2+, but is only 80% pure as judged by analytical disc gel electrophoresis. The purified enzyme is unstable but can be stabilized by addition of Ca2+ and the protein modulator; this is in contrast to the less pure preparations of Ca2+-activatable phosphodiesterase which are destabilized by the protein modulator in the presence of Ca2+.     

Inhibition of Ca2+-activated cyclic nucleotide phosphodiesterase reaction by a heat-stable inhibitor protein from bovine brain.

An inhibitor protein of cyclic nucleotide phosphodiesterase is demonstrated in bovine brain extract and separated from modulator binding protein, a recently discovered inhibitory factor of phosphodiesterase. The new inhibitor protein is similar to the cyclic AMP phosphodiesterase inhibitor from bovine retina (Dumler, I. L., and Etingof, F. N. 1976) Biochim. Biophys, Acta 429, 474-484) in its heat stability: it retains full activity upon heating in a boiling water bath for 2 min. The new inhibitor protein counteracts the activation of the Ca2+-activatable cyclic nucleotide phosphodiesterase by the Ca2+-dependent modulator protein without affecting the basal activity of the enzyme. The inhibition of phosphodiesterase by the inhibitor can be reversed by high concentrations of modulator protein but is not influenced by a 20-fold increase in Ca2+ concentration. In contrast, a Ca2+-independent form of cyclic nucleotide phosphodiesterase is not inhibited by the inhibitor protein. These results suggest that the heat-stable inhibitor protein is specific against the action of the Ca2+-dependent modulator protein. Gel filtration analyses on Sephadex G-75 and G-100 columns have shown that the inhibitor protein and the modulator protein may associate in the presence of Ca2+. The molecular weights determined by the gel filtration for the free inhibitor protein and the complex of the inhibitor and modulator protein are about 70,000 and 85,000, respectively.     

Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys.

The activities of myometrial cyclic nucleotide phosphodiesterases (PDEs) and the sensitivity of these enzymes to the effector molecules, cGMP and cAMP, were determined in the 100,000 g supernatant of homogenates from pregnant and spayed rhesus monkeys. The specific activities (per mg nitrogen) of the myometrial cyclic nucleotide PDEs in the supernatant from spayed monkeys were higher than those from pregnant monkeys at all substrate levels studied. However, when calculated on the basis of the DNA content of the myometrium, which was 8 times higher in the spayed than in the pregnant animals, the specific activities were lower in the tissue from spayed animals. At substrate levels of 2 . 5 micron-cAMP, low levels of cGMP (0 . 1-1 . 0 micron) caused the same percentage increase in cGMP-PDE activity in both tissues. At high substrate levels of 100 micron-cAMP, 1 micron-cGMP inhibited only the cAMP-PDE from spayed monkeys, and the enzyme from spayed monkeys was more effectively inhibited by 10 and 40 micron-cGMP than was the enzyme from pregnant animals. The cGMP-PDE activity was inhibited by cAMP (1 . 0-50 . 0 micron), and the percentage inhibition with increasing levels of cAMP appeared to be similar in the two series. The levels of cGMP and cAMP that modify the rate of hydrolysis of the other nucleotide in rhesus myometrium seem to be within the physiological range for these compounds in situ. It therefore appears possible that cAMP and cGMP are each involved in regulating the degradation of the other nucleotide in rhesus myometrium.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

Ca2+ or Mg2+ nucleotide phosphohydrolases in myometrium: two ecto-enzymes.

A high level of Ca2+ or Mg2+ nucleotide phosphohydrolase activity is present on the outside surface of intact myometrial cells and is also observed in the isolated plasma membranes. About half of this activity is labile while the remainder is stable. The characteristics of the activities suggest the presence of at least two different ecto-enzymes. The stable component (Km for Ca2+ about 0.1 mM) accepts XTP or XDP as substrate, is not inhibited by p-chloromercuriphenylsulfonate or inorganic phosphate, but is inhibited by 20 mM NaN3. The labile component (Km for Ca2+ nearly 1 mM) cleaves XTP but not XDP, and is inhibited by p-chloromercuriphenyl-sulfonate and inorganic phosphate, but not by NaN3. The activity of the labile component can be restored by removing the cells from the incubation medium and resuspending them in fresh medium. This suggests that the 'lability' is due to product inhibition, probably by inorganic orthophosphate. While the Ca2+ pump of myometrial plasma membranes was inhibited by 0.1 microM oxytocin, these ecto-enzymes were unaffected by oxytocin concentrations up to 10 microM. Because of its high activity and rapid inactivation by product inhibition, the labile enzyme may be involved in the regulation of purinergic receptors.     

Purification of the heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase by affinity chromatography.

The recently discovered heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase (Sharma, R. K., Wirch, E. & Warg, J. H. (1978) J. Biol. Chem., in press) has been purified 238 214-fold from bovine brain extract using an affinity column of the modulator protein--Sepharose 4B conjugate. The purified sample appears to be homogeneous as judged by sodium dodecyl sulphate (SDS) gel electrophoresis. The protein band has a mobility corresponding to that of a polypeptide of molecular weight 68 000. Since the heat-stable inhibitor protein has a molecular weight of 70 000 under nondenaturing conditions, it suggests that it is a monomeric protein. The protein has no inhibitory activity toward the cAMP-dependent protein kinase or protein phosphatase. The purified sample has been tested for various enzyme activities which include ATPase, GTPase, cAMP phosphodiesterase, cGMP phosphodiesterase, 5'-nucleotidase, and protein kinase. None of these activities are exhibited by the purified sample.     

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.     

Direct interaction of calmodulin antagonists with Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase

Trypsin-treated Ca2+/calmodulin-dependent phosphodiesterase (CA2+-PDE), which had lost its sensitivity to Ca2+-calmodulin, was inhibited by various calmodulin antagonists, trifluoperazine, chlorpromazine, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and aminoalkyl chain analogues of W-7 (A-3, A-4, A-5, I-240, A-6, A-7). These inhibitory effects were less than those on calmodulin-activated Ca2+-PDE. The ability of these compounds to inhibit trypsin-treated Ca2+-PDE correlated well with the inhibitory effect on calmodulin-activated Ca2+-PDE. W-7 inhibited trypsin-treated Ca2+-PDE in a competitive fashion with respect to cyclic GMP and the Ki value was 300 microM. The inhibition of trypsin-treated Ca2+-PDE by W-7 (300 microM) or A-7 (100 microM) was overcome by the addition of excess calmodulin. Trypsin-treated Ca2+-PDE can bind to W-7-coupled cyanogen bromide-activated Sepharose 4B in the presence of 1 mM EGTA. These results suggest that Ca2+-PDE possesses a binding site for calmodulin antagonists and that the binding site for these antagonists on this enzyme may be structurally similar to the binding site on calmodulin itself.     

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurosproa crassa

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

Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line.

This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.     

Interaction of 125I-labeled Ca2+-dependent regulator protein with cyclic nucleotide phosphodiesterase and its inhibitory protein.

The Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase was labeled with 125I to the extent of 1 mol of monoiodotyrosine per mol. The iodinated protein showed a small decrease in affinity for phosphodiesterase but gave the same maximal level of activation of the enzyme as did the unmodified regulator protein. Iodinated regulator protein formed complexes with both highly purified cyclic nucleotide phosphodiesterase and phosphodiesterase inhibitory protein in the presence but not in the absence of Ca2+ as demonstrated by ultracentrifugation in glycerol gradients. Cross-linking experiments indicate that the Ca2+-dependent regulator protein interacts with the large subunit of the inhibitory protein.