Calmodulin-dependent Cyclic Nucleotide Phosphodiesterase Research Articles

Cyclic nucleotide signaling in polycystic kidney disease

Increased levels of 3'-5'-cyclic adenosine monophosphate (cAMP) stimulate cell proliferation and fluid secretion in polycystic kidney disease. Levels of this molecule are more sensitive to inhibition of phosphodiesterases (PDEs), whose activity far exceeds the rate of cAMP synthesis by adenylyl cyclase. Several PDEs exist, and here we measured the activity and expression of PDE families, their isoforms, and the expression of downstream effectors of cAMP signaling in the kidneys of rodents with polycystic kidney disease. We found a higher overall PDE activity in kidneys from mice as compared with rats, as well as a higher contribution of PDE1, relative to PDE4 and PDE3, to total PDE activity of kidney lysates and lower PDE1, PDE3, and PDE4 activities in the kidneys of cystic as compared with wild-type mice. There were reduced amounts of several PDE1, PDE3, and PDE4 proteins, possibly due to increased protein degradation despite an upregulation of their mRNA. Increased levels of cGMP were found in the kidneys of cystic animals, suggesting in vivo downregulation of PDE1 activity. We found an additive stimulatory effect of cAMP and cGMP on cystogenesis in vitro. Cyclic AMP-dependent protein kinase subunits Ialpha and IIbeta, PKare, the transcription factor CREB-1 mRNA, and CREM, ATF-1, and ICER proteins were upregulated in the kidneys of cystic as compared with wild-type animals. Our study suggests that alterations in cyclic nucleotide catabolism may render cystic epithelium particularly susceptible to factors acting on Gs-coupled receptors. This may account, in part, for increased cyclic nucleotide signaling in polycystic kidney disease and contribute substantially to disease progression.

Surface plasmon resonance characterization of calspermin–calmodulin binding kinetics

We cloned, expressed, and purified a chimeric fusion between a soluble green fluorescent protein (smGFP) and the calmodulin binding protein calspermin. We have shown that the fusion protein, labeled smGN, has a Ki in the calmodulin-dependent cyclic nucleotide phosphodiesterase activity assay of 1.97nM, i.e., 3800 times smaller than that of the commonly used calmodulin inhibitor W7. Association and dissociation rate constants (ka and kd) and the dissociation equilibrium constant (KD) of smGN for calmodulin were determined using surface plasmon resonance (SPR). The ka=1.24·106M−1s−1, the kd=5.49·10−3s−1, and the KD=4.42·10−9M. We also found that the GFP moiety was important for successfully binding calspermin to the surface of the CM5 flow cell at a sufficiently high concentration for SPR, and that this procedure may be used for SPR analysis of other acidic polypeptides, whose pI⩽4. To determine whether smGN might also bind to other calmodulin-like proteins in a heterologous system, we purified proteins from a plant total cell extract or a plant total protein extract by affinity chromatography against smGN. The purified proteins were identified as calmodulins by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and liquid chromatography–tandem mass spectrometry, indicating a high level of specificity. We conclude that the high affinity and specific binding between smGN and calmodulin make it an easily localized recombinant alternative to chemical calmodulin inhibitors.

Cloning of the Gene Cluster Responsible for Biosynthesis of KS-505a (Longestin), a Unique Tetraterpenoid

KS-505a (longestin), produced by Streptomyces argenteolus, has a unique structure that consists of a tetraterpene (C40) skeleton, to which a 2-O-methylglucuronic acid and an o-succinyl benzoate moiety are attached. It is a novel inhibitor of calmodulin-dependent cyclic-nucleotide phosphodiesterase, which is representative of a potent anti-amnesia drug. As a first step to understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we cloned a KS505a biosynthetic gene cluster. First we searched for a gene encoding octaprenyl diphosphates, which yielded a C40 precursor by PCR, and four candidate genes were obtained. Among these, one was confirmed to have the expected enzyme activity by recombinant enzyme assay. On the basis of an analysis of the flanking regions of the gene, a putative KS-505a biosynthetic gene cluster consisting of 24 ORFs was judged perhaps to be present on a 28-kb DNA fragment. A gene disruption experiment was also employed to confirm that the cluster indeed participated in KS-505a biosynthesis. This is believed to be the first report detailing the gene cluster of a cyclized tetraterpenoid.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) splice variants from bovine cardiac muscle

Calmodulin-dependent cyclic nucleotide phopshodiesterase (PDE1) has been extensively characterized and is a key enzyme involved in the complex interaction between cyclic nucleotide and Ca 2+ second-messenger systems. It is well established that PDE1 exists in different isozymes. For example, bovine brain tissue has two PDE1 isozymes (PDE1A2 and PDE1B1) whereas only one form (PDE1A1) is reported in bovine cardiac tissue. In this study, we report the cloning of two cDNA splice variants of PDE1: PDE1-small and PDE1-large, from bovine cardiac tissue. Their amino acid sequence similarity to PDE1 sequences from other mammalian species showed that all are very conserved, suggesting their importance in cellular functions. Interestingly, compared to other mammalian species, bovine PDE1A, PDE-small and PDE-large show a deletion at the C-terminal end of the catalytic domain of the gene. Although the significance of this deletion at this crucial location of the gene is not known, we have successfully over-expressed both PDE1-small and PDE1-large splice variants in E. coli and these splice variants are characterized in terms of Western blot, biotinylated calmodulin overlay and peptide mass fingerprinting. Results from these studies suggested that these two splice variants belong to the PDE1 superfamily. To our knowledge, this is the first report on cloning and characterization of these cDNA variants from bovine cardiac tissue . Since there are at least two isoforms of PDE1 in bovine heart tissue, this merits further in-depth study.

Involvement of GSK-3β and DYRK1B in Differentiation-inducing Factor-3-induced Phosphorylation of Cyclin D1 in HeLa Cells

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr(286) and Thr(288) were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr(286) and/or Thr(288) prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.

Gene expression and protein localization of calmodulin‐dependent phosphodiesterase in adult rat retina

Calcium calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) was identified in crude extract and immunolabeled sections of rat retina. Both cAMP and cGMP PDE activities were stimulated by calcium-calmodulin (4.7-fold and 2.3-fold, respectively). To characterize PDE1 isoforms in retinal cells further, we used antibodies that specifically recognize PDE1 gene products. PDE1B antibody stained a band at molecular mass of 63 kDa whereas PDE1C antibody recognized two bands at 74- and 70-kDa molecular masses. Two PDE1A antibodies (against N-terminal and C-terminal peptides) detected a band at 79 kDa never described before. Immunohistochemical analysis showed a distribution of PDE1A in the outer retina with a bright fluorescence in the outer segments of photoreceptors. PDE1B is uniformly distributed across the retina. PDE1C is confined mainly to the inner retina, with a precise localization in the inner nuclear layer. Immunostaining with choline acetyltransferase antibody indicates localization in cholinergic amacrine cell. The present data provide evidence of expression of PDE1 isoforms in mammalian retina with a complementary distribution of PDE1A and PDE1C, suggesting different roles in retinal function.

Ca2+/calmodulin-dependent cyclic nucleotide phosphodiesterase in cGMP metabolism in rabbit parotid acinar cells

Muscarinic cholinergic receptor activation provokes cGMP formation in parotid acinar cells. We investigated the involvement of Ca(2+)/calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) in cGMP breakdown in rabbit parotid acinar cells. The muscarinic agonist carbachol stimulated cGMP formation in the cells. The carbachol-induced cGMP formation was enhanced in the presence of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MM-IBMX), a PDE1 inhibitor. cGMPPDE activity in rabbit parotid acinar cells was reduced by about 25% in the absence of Ca(2+)/ calmodulin or in the presence of MM-IBMX. Ca(2+)/calmodulin-dependent cGMP-PDE in rabbit parotid acinar cells was purified using Calmodulin-Sepharose 4B and Mono Q ion-exchange column chromatography. Two dominant fractions with cGMP-PDE activity, referred to as the P-1 and P-2 fractions, were eluted from the Mono Q ion-exchange column. The Km values for cGMP of PDE in the P-1 and P-2 fractions were 0.82 microM and 0.40 microM, respectively, which were much lower than that for cAMP. The EC(50) for Ca(2+) and calmodulin of PDEs in the P-1 and P-2 fractions were 458 nM and 426 nM, respectively, and 32 nM and 137 nM, respectively. Protein bands that crossreacted with anti-PDE1A antibody were detected. These results suggest that Ca(2+)/calmodulin-dependent PDE, PDE1A, is involved in cGMP breakdown in rabbit parotid acinar cells.

Effect of selected phytotoxins from Guanomyces polythrix on the calmodulin-dependent activity of the enzymes cAMP phosphodiesterase and NAD-kinase.

The effect of a series of phytotoxins isolated from the fungus Guanomyces polytrix on calmodulin (CaM)-dependent nicotinamide adenine dinucleotide kinase (NADK) and CaM-dependent cyclic nucleotide phosphodiesterase (PDE) activities was investigated. The results indicated that (2S,3S)-5-hydroxy-6,8-dimethoxy-2,3-dimethyl-4H-2,3-dihydronaphtho[2,3-b]-pyran-4-one, (2S,3S)-5-hydroxy-6,8,10-trimethoxy-2,3-dimethyl-4H-2,3-dihydro-naphtho[2,3-b]-pyran-4-one, (2S,3R)-5-hydroxy-6,8-dimethoxy-2,3-dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one, (2S,3R)-5-hydroxy-6,8,10-trime-thoxy-2,3-dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one, 5-hydro-xy-6,8-dimethoxy-2,3-dimethyl-4H-naphtho[2,3-b]-pyran-4-one, rubrofusarin B, and ergosta-4,6,8(14),22-tetraen-3-one inhibited the activation of both target enzymes in the presence of CaM. On the other hand, (2S)-5-hydroxy-6,8-dimethoxy-2-methyl-4H-2,3-dihydronaphtho[2,3-b]-pyran-4-one and (2S)-5-hydroxy-6,8,10-trimethoxy-2-methyl-4H-2,3-dihydronaphtho-[2,3-b]-pyran-4-one inhibited the activation of PDE and the basal activity of NADK. Thus, these phytotoxins are CaM inhibitors and may exert their phytotoxic action by inhibiting the CaM-dependent process, although they could also interfere with other cellular metabolic phenomena. This is the first report of the use of the NADK assay to detect or quantify CaM inhibitors, and it could be a valuable tool for studying those CaM isoforms regulating NADK.

Calmodulin-dependent cyclic nucleotide phosphodiesterase activity is altered by 20 microT magnetostatic fields.

Absorbance measurements at 660 nm of calmodulin (CaM) dependent cyclic nucleotide phosphodiesterase activity under cell free conditions indicate that 30-min exposures to weak magnetostatic field intensities alters this activity, compared to zero magnetic field exposures. This effect depends nonlinearly on the concentration of free calcium, with maximum magnetic interaction apparently occurring at an optimal Ca(2+) concentration corresponding to 50% activation (EC(50)). If one regards Ca(2+)/CaM activation as a switching process, then increasing the magnetic field at Ca(2+) levels in excess of optimal acts to bias this switch towards lower calcium concentrations. A magnetic dependence has been previously reported by others in an homologous system, CaM dependent myosin light chain phosphorylation, implying that there may be an underlying magnetic interaction that involves the initial Ca(2+)/CaM binding process common to both enzymatic pathways. The level of magnetostatic intensity at which this effect is observed ( approximately 20 microT) implies that CaM activation may be functionally sensitive to the geomagnetic field.

Localization and regulation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase by cyclic AMP-dependent protein kinase

Calmodulin-dependent cyclic nucleotide phosphodiesterase is one of the key enzymes involved in the complex interactions, which occur between the cyclic nucleotide and Ca2+ second-messenger systems. In eye, cAMP regulation is important in a variety of physiological processes such as aqueous humor regulation, photoreceptor signal transduction and retinal blood flow. Bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was purified to apparent homogeneity and the isolated enzyme had a significantly higher affinity for calmodulin and Ca2+. Immunohistology revealed calmodulin-dependent cyclic nucleotide phospho-diesterase expression in corneal epithelium, retina and optic nerve of the eye. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase and the following observations were made. Firstly, the phosphorylation resulted in the incorporation of 1 mol of phosphate per mol of subunit, resulting in higher calmodulin and Ca2+ concentration requirement for calmodulin-dependent cyclic nucleotide phosphodiesterase activation. Secondly, Ca2+ and calmodulin prevented the phosphorylation. Thirdly, the phosphorylation of calmodulin-dependent cyclic nucleotide phosphodiesterase could be reversed by the calmodulin-dependent phosphatase, calcineurin. Analysis of the complex regulatory properties of the calmodulin-dependent cyclic nucleotide phosphodiesterase in the eye has led to the suggestion that fluxes of cAMP and Ca2+ during cell activation are closely coupled and that calmodulin-dependent cyclic nucleotide phosphodiesterase plays a key role in this signal coupling phenomenon.

Calmodulin-dependent cyclic nucleotide phosphodiesterase in an experimental rat model of cardiac ischemia-reperfusion.

In the present study, we investigated the activity and expression of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) and the effects of calpains in rat heart after ischemia and reperfusion. Immunohistochemical studies indicated that CaMPDE in normal heart is localized in myocardial cells. Rat ischemic heart showed a decrease in CaMPDE activity in the presence of Ca2+ and calmodulin; however, in ischemic-reperfusion tissue a progressive increase in Ca2+ and calmodulin-independent cyclic nucleotide phosphodiesterase (CaM-independent PDE) activity was observed. Perfusion of hearts with cell-permeable calpain inhibitor suppressed the increase of Ca2+ and CaM-independent PDE activity. Protein expression of CaMPDE was uneffected by hypoxic injury to rat myocardium. The purified heart CaMPDE was proteolyzed by calpains into a 45 kDa immunoreactive fragment in vitro. Based on these results, we propose that hypoxic injury to rat myocardium results in the generation of CaM-independent PDE by calpain mediated proteolysis, allowing the maintenance of cAMP concentrations within the physiological range.

A method for the purification of phospho(Tyr)calmodulin free of nonphosphorylated calmodulin.

Phosphocalmodulin has been shown to have a differential biological activity compared to nonphosphorylated calmodulin when assayed on a variety of calmodulin-dependent systems. However, the phosphocalmodulin preparations used so far in those experiments were not necessarily free of nonphosphorylated calmodulin. Therefore, the results obtained may not unquestionably show the real effect of pure phosphocalmodulin on the systems under study. To solve this problem, we describe here a method for the purification of phospho(Tyr)calmodulin free of nonphosphorylated calmodulin. The procedure consists of the following steps: (i) phosphorylation of calmodulin by a fraction enriched in epidermal growth factor receptor tyrosine kinase from rat liver isolated by calmodulin affinity chromatography, (ii) isolation of a calmodulin/phosphocalmodulin mixture by Ca(2+)-dependent chromatography in phenyl-Sepharose, (iii) purification of phospho(Tyr)calmodulin using an anti-phosphotyrosine antibody immobilized in agarose upon elution with phenyl phosphate, and (iv) removal of phenyl phosphate from the phospho(Tyr)calmodulin preparation by filtration chromatography in a Bio-Gel P-2 column. The obtained phospho(Tyr)calmodulin preparation was highly pure and essentially free of nonphosphorylated calmodulin because of the use of anti-phosphotyrosine affinity chromatography. We demonstrate that this ultrapure phospho(Tyr)calmodulin preparation is totally incapable of activating the calmodulin-dependent cyclic nucleotide phosphodiesterase. In contrast, when a nonpurified phospho(Tyr)calmodulin preparation was used a partial activation of this enzyme was observed.

In VitroGeneration of an Active Calmodulin-Independent Phosphodiesterase from Brain Calmodulin-Dependent Phosphodiesterase (PDE1A2) by m-Calpain

In the present study we have shown that bovine brain 60-kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme (CaMPDE - PDE1A2) is proteolyzed by a Ca2+-dependent cysteine protease, m-calpain. The proteolysis of PDE1A2 by m-calpain results in its conversion to a totally calmodulin (CaM)-independent form accompanied by degradation of PDE1A2 into a 45-kDa catalytic fragment and a 15-kDa fragment. The activity of PDE1A2 is unaffected by the presence or absence of CaM during cleavage, suggesting that the interaction between CaM and PDE1A2 does not alter substrate recognition by calpain. Furthermore, we provide evidence, based on the studies of CaM overlay and phosphorylation, that the cleavage site is not present either in the CaM-binding domain or phosphorylation site. N-terminal sequence analysis of the 45-kDa fragment indicated that cleavage occurs between residues126Gln and127Ala, and eliminates the CaM-dependent activity of carboxy termini PDE1A2. The present findings suggest that limited proteolysis in the brain through calpains could be an alternate mechanism for activating CaMPDE(s) and for regulating intracellular levels of cAMP.

Novel protein inhibitor of calmodulin-dependent cyclic nucleotide phosphodiesterase from glioblastoma multiforme.

Previous investigations from our laboratory have demonstrated a significant reduction in the catalytic function of the 60 kDa and 63 kDa isozymes of calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) when comparing human cerebral tissue that was free of tumor and glioblastoma multiforme (GBM) and gliosarcoma [Lal S., Raju R. V. S., Macaulay R. B. J., and Sharma R. K. (1996) Can. J. Neurol. Sci., 23, 245-250]. The results suggested the possibility of an endogenously produced inhibitor of CaMPDE expressed in these tumors. Further investigation has initially characterized the presence of a heat-labile, protein inhibitor of both the 60 kDa and 63 kDa isozymes of CaMPDE. Sephacryl S-200 gel filtration column chromatography indicated that the inhibitor has an apparent molecular weight of 22 kDa and experimental evidence demonstrates that this inhibitor protein may act independently of calmodulin, and is therefore a novel CaMPDE inhibitor. Previous work on non-CNS tumors has shown high levels of CaMPDE activity and absence of an inhibitor. This suggests that a different mechanism may exist for the proliferation of these subsets of tumors.

Human erythrocytes contain Ca2+, calmodulin-dependent cyclic nucleotide phosphodiesterase which is involved in the hydrolysis of cGMP.

To determine whether phosphodiesterase (PDE) is involved in the degradation of cGMP in human erythrocytes, we studied the cell cGMP content in the presence of different PDE inhibitors: zaprinast and dipyridamole, specific inhibitors of cGMP-binding, cGMP-specific PDE (cG-BPDE); vinpocetine, a specific inhibitor of Ca2+, calmodulin-dependent phosphodiesterase (CaM-PDE); an unspecific inhibitor, 3-isobutyl-1-methylxanthine (IBMX). IBMX, zaprinast, and dipyridamole at 30 microM did not affect the intracellular cGMP content. However, vinpocetine at this concentration increased the cGMP content by 102 +/- 14% (p < 0.05). The effect of vinpocetine was dose-dependent, reached the maximal level after 1 min of incubation and flattened at the same level. Ca2+ (10 microM) in the presence of the Ca(2+)-ionophore, A23187 (5 microM), decreased the cGMP content (-23% +/- 4; p < 0.05), which can be explained by the CaM-PDE activation. The Ca(2+)-induced decrease in cGMP was completely inhibited by the CaM antagonist, W-7 (100 microM). These data suggest that erythrocytes contain Ca2+, CaM-PDE.

Differential effects of tamoxifen-like compounds on osteoclastic bone degradation, H+-ATPase activity, calmodulin-dependent cyclic nucleotide phosphodiesterase activity, and calmodulin binding

We studied effects of calmodulin antagonists on osteoclastic activity and calmodulin-dependent HCl transport. The results were compared to effects on the calmodulin-dependent phosphodiesterase and antagonist-calmodulin binding affinity. Avian osteoclast degradation of labeled bone was inhibited approximately 40% by trifluoperazine or tamoxifen with half-maximal effects at 1-3 microM. Four benzopyrans structurally resembling tamoxifen were compared: d-centchroman inhibited resorption 30%, with half-maximal effect at approximately 100 nM, cischroman and CDRI 85/287 gave 15-20% inhibition, and l-centchroman was ineffective. No benzopyran inhibited cell attachment or protein synthesis below 10 microM. However, ATP-dependent membrane vesicle acridine transport showed that H(+)-ATPase activity was abolished by all compounds with 50% effects at 0.25-1 microM. All compounds also inhibited calmodulin-dependent cyclic nucleotide phosphodiesterase at micromolar calcium. Relative potency varied with assay type, but d- and l-centchroman, surprisingly, inhibited both H(+)-ATPase and phosphodiesterase activity at similar concentrations. However, d- and l-centchroman effects in either assay diverged at nanomolar calcium. Of benzopyrans tested, only the d-centchroman effects were calcium-dependent. Interaction of compounds with calmodulin at similar concentrations were confirmed by displacement of labeled calmodulin from immobilized trifluoperazine. Thus, the compounds tested all interact with calmodulin directly to varying degrees, and the observed osteoclast inhibition is consistent with calmodulin-mediated effects. However, calmodulin antagonist activity varies between specific reactions, and free calcium regulates specificity of some interactions. Effects on whole cells probably also reflect other properties, including transport into cells.

Mechanisms of inhibition of calmodulin-stimulated cyclic nucleotide phosphodiesterase by dihydropyridine calcium antagonists.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interaction between the cyclic nucleotide and Ca2+ second-messenger systems. CaMPDE exists as tissue-specific isozymes, and initially these isozymes were designated according to their respective subunit molecular mass. A variety of pharmacological agents have been used to inhibit CaMPDE, and this inhibition occurs mostly via Ca2+-dependent association with the proteins. We have examined the effect of dihydropyridine Ca2+-channel blockers felodipine and nicardipine on CaMPDE. The results suggest that the 63-kDa (PDE 1B1) and 60-kDa (PDE 1A2) CaMPDE isozymes are inhibited by felodipine and nicardipine by partial competitive inhibition and that these two Ca2+ antagonists appear to counteract each other. This study further demonstrates the existence of a specific site, distinct from the active site on CaMPDE, that exhibits high-affinity binding of these drugs. Felodipine and nicardipine have similar affinities for 60-kDa CaMPDE isozymes but bring about different levels of enzyme inhibition, suggesting the possibility of designing specific drugs that can protect the enzyme from inhibition by dihydropyridine Ca2+-channel blockers.

Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin.

The binding of Mg2+ to calmodulin (CaM) and the effect of Mg2+ on the binding of Ca2+-CaM to target peptides were examined using two-dimensional nuclear magnetic resonance and fluorescence spectroscopic techniques. We found that Mg2+ preferentially binds to Ca2+-binding sites I and IV of CaM in the absence of Ca2+ and that Ca2+-binding site III displays the lowest affinity for Mg2+. In contrast to the marked structural transitions induced by Ca2+ binding, Mg2+ binding causes only localized conformational changes within the four Ca2+-binding loops of CaM. Therefore, Mg2+ does not seem to be able to cause significant structural effects required for the interaction of CaM with target proteins. The presence of excess Mg2+ (up to 10 mM) does not change the order and cooperativity of Ca2+ binding to CaM, and as expected, the structure of Ca2+-saturated CaM is not affected by the presence of Mg2+. However, we found that the binding of Ca2+-saturated CaM to target peptides is affected by Mg2+ with the binding affinity decreasing as the Mg2+ concentration increases. Three different peptides, corresponding to the CaM binding domain of skeletal muscle myosin light-chain kinase (MLCK), CaM-dependent cyclic nucleotide phosphodiesterase (PDE), and smooth muscle caldesmon (CaD), were examined and show different reductions in their affinities toward CaM. The CaM-binding affinity of the MLCK peptide in the presence of 50 mM Mg2+ is approximately 40-fold lower than that seen in the absence of Mg2+, and a similar response was observed for the PDE peptide. The affinity of the CaD peptide for CaM also shows a Mg2+ dependence, though to a much lower magnitude. The Mg2+-dependent decrease in the affinities between CaM and its target peptides is an intrinsic property of Mg2+ rather than a nonspecific ionic effect, as other metal ions such as Na+ do not completely replicate the effect of Mg2+. The inhibitory effect of Mg2+ on the formation of complexes between CaM and its targets may contribute to the specificity of CaM in target activation in response to cellular Ca2+ concentration fluctuations.

Calmodulin-Dependent Cyclic Nucleotide Phosphodiesterase from Bovine Eye: High Calmodulin Affinity Isozyme Immunologically Related to the Brain 60-kDa Isozyme

Calmodulin-dependent cyclic nucleotide phosphodiesterase was identified in and purified to apparent homogeneity from the total calmodulin-binding protein fraction of bovine eye in a single step by immunoaffinity chromatography. The bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase is immunologically similar to the bovine brain 60-kDa isozyme. The purified enzyme had higher affinity for calmodulin than the 60-kDa phosphodiesterase isozyme from bovine brain, but similar affinity to that of the heart isozyme. When the Ca 2+-dependence of the eye enzyme was compared to cardiac calmodulin-dependent cyclic nucleotide phosphodiesterase at an identical concentration of calmodulin, the bovine eye calmodulin-dependent cyclic nucleotide phosphodiesterase was activated at the same Ca 2+concentration as the bovine heart calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme.

Amantadine: an antiparkinsonian agent inhibits bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme

The effect of amantadine (an antiparkinsonian agent) on calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes was investigated. Amantadine inhibited bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase but not the bovine brain 63 kDa, heart and lung calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes. The inhibition of bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase was overcome by increasing the concentration of calmodulin. This suggests that amantadine may be an antagonist of calmodulin or act specifically and reversibly on the action of calmodulin. The bovine brain 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme is predominantly expressed in the brain and its inhibition may result in increased intracellular levels of cyclic AMP (cAMP). The increased intracellular levels of cAMP have a protective role for dopaminergic neurons. The present findings suggest that amantadine may be a valuable tool to investigate the physiological role of 60 kDa calmodulin-dependent cyclic nucleotide phosphodiesterase isozyme in the progression of Parkinson's disease and gives a new insight into the action of this drug.