Brain Cyclic Nucleotide Phosphodiesterase Research Articles

Structural Evidence That Brain Cyclic Nucleotide Phosphodiesterase Is a Member of the 2H Phosphodiesterase Superfamily

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP) is an enzyme abundantly present in the central nervous system of mammals and some vertebrates. In vitro, CNP specifically catalyzes the hydrolysis of 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the physiologically relevant in vivo substrate remains obscure. Here, we report the medium resolution NMR structure of the catalytic domain of rat CNP with phosphate bound and describe its binding to CNP inhibitors. The structure has a bilobal arrangement of two modules, each consisting of a four-stranded beta-sheet and two alpha-helices. The beta-sheets form a large cavity containing a number of positively charged and aromatic residues. The structure is similar to those of the cyclic phosphodiesterase from Arabidopsis thaliana and the 2'-5' RNA ligase from Thermus thermophilus, placing CNP in the superfamily of 2H phosphodiesterases that contain two tetrapeptide HX(T/S)X motifs. NMR titrations of the CNP catalytic domain with inhibitors and kinetic studies of site-directed mutants reveal a protein conformational change that occurs upon binding.

Neurite formation in NG108-15 neuroblastoma x glioma hybrid cells by KS-505a, a potent inhibitor of brain cyclic nucleotide phosphodiesterase.

Neurite formation in NG108-15 neuroblastoma x glioma hybrid cells by KS-505a, a potent inhibitor of brain cyclic nucleotide phosphodiesterase.

A test to evaluate the effect of individual components of ethylene glycol bis(β-aminoethyl ether)- N,N,N′,N′-tetraacetic acid buffers on enzymatic activity

A simple dilution test for evaluating the individual effect on enzymatic activity of [Ca 2+], [EGTA], or [Ca·EGTA] variations in Ca-EGTA buffers is presented. We verified that a 50-fold dilution of the buffer (25-0.5 m m) at constant pH did not affect [Ca 2+] (measured with fura-2), whereas [EGTA] and [Ca·EGTA] varied. Therefore the test can be applied to evaluate the proper effect of Ca 2+ in a Ca-EGTA buffer on enzyme activity because such an effect is expected to remain unchanged upon dilution of the buffer. Applications of the test are shown for three enzymes apparently sensitive to Ca 2+ but found to be effectively influenced only by Ca·EGTA (liver glucose-6-phosphatase), EGTA (intestinal mucosa phosphatase), or indeed Ca 2+ (brain cyclic nucleotide phosphodiesterase).

Retinoic acid inhibits calmodulin binding to human erythrocyte membranes and reduces membrane Ca2(+)-adenosine triphosphatase activity.

Ca2(+)-ATPase activity in human red cell membranes is dependent on the presence of calmodulin. All trans-retinoic acid inhibited human red cell membrane Ca2(+)-ATPase activity in vitro in a concentration-dependent manner (10(-8) to 10(-4) M). In contrast, retinol, retinal, 13-cis-retinoic acid and the benzene ring analogue of retinoic acid did not alter enzyme activity. Purified calmodulin (up to 500 ng/ml, 3 X 10(-8) M) added to red cell membranes, in the presence of inhibitory concentrations of retinoic acid, only partially restored Ca2(+)-ATPase activity. 125I-Calmodulin bound to red cell membranes was displaced by unlabeled retinoic acid (50% reduction at 10(-8) M retinoic acid), as effectively as by unlabeled calmodulin. Another calmodulin-stimulable enzyme, bovine brain cyclic nucleotide phosphodiesterase, was unaffected by retinoic acid. 8-Anilino-1-naphthalene sulfonic acid bound to calmodulin, studied spectrofluorometrically, was not displaced by retinoic acid. Thus, retinoic acid inhibits calmodulin binding to red cell membranes, reducing calmodulin-stimulable Ca2(+)-ATPase activity. Retinoic acid does not directly interact with calmodulin, but rather exerts its effect by interfering with calmodulin access to the membrane enzyme. These effects occur at physiological concentrations of the retinoid.

Interaction of amiodarone and its analogs with calmodulin

Benzofurans have important actions on the electrical properties of myocardium; the biochemical basis of those actions is not known. Crystallographic examination of these compounds has revealed that benzofurans share structural homologies with the traditional calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalene and trifluoperazine. In the present study, the ability of amiodarone, desethylamiodarone, and benziodarone to displace the fluorescent ligand 8-anilino-1-napthalene sulfonic acid (ANS) from calmodulin, to modulate the fluorescence emission of dansylcalmodulin, and to inhibit the activation by calmodulin of bovine brain cyclic nucleotide phosphodiesterase and human erythrocyte membrane Ca 2+-ATPase were investigated at concentrations ranging from 10 −8 to 10 −6 m. These benzofurans displaced ANS from calmodulin with nearly equal efficiency upon forming a 1:1 complex with that protein. Each of these compounds also produced a decreased fluorescence emission of dansylcalmodulin, but with relative efficiencies being desethylamiodarone > amiodarone > benziodarone. Amiodarone and desethylamiodarone inhibited calmodulin-stimulable phosphodiesterase activity with similar potencies. Amiodarone and benziodarone inhibited calmodulin-stimulable Ca 2+-ATPase activity equally, but desethylamiodarone had no effect. The observed differential effects of the amiodarone analogs suggest that calmodulin may possess multiple benzofuran-binding sites that are recognized by specific targets and ligands of this Ca 2+-binding protein and that the cellular action of amiodarone and its analogs may reflect calmodulin antagonism.

Characterization of specific fluorenylmethyloxycarbonyl-containing calmodulin adducts by spectroscopy and phosphodiesterase stimulation

A reagent (I, N4-(9′-fluorenylmethyloxycarbonyl-4-amino-1-oxyl-4-succinimidyloxycarbonyl-2,2,6,6-tetramethylpiperidine)) that acylates calmodulin specifically at lysines 75 and 148 was recently described (Jackson and Puett, 1984). Chromatographic procedures are described that permit purification to apparent homogeneity of a 1 : 1 and a 2 : 1 adduct characterized by modification at just Lys 75 or at Lys 75 and Lys 148, respectively. These adducts are suitable for detailed characterization in an effort to provide information on calmodulin structure-function relationships. The adducts were incapable of, or exhibited low potency (e.g., 0.1% that of calmodulin) in, stimulating the activity of an activatable bovine brain cyclic nucleotide phosphodiesterase (3′,5′-cyclic AMP 5′-nucleotidehydrolase, EC 3.1.4.17) preparation. Electron paramagnetic resonance (EPR) spectroscopy of the adducts yielded rotational correlation times of approximately 3–6 nsec, in agreement with the expected value for a hydrated protein of this molecular weight (∼5–7 nsec). Thus, the nitroxide reporter group appears to monitor closely the motion of the protein, and there is no evidence of a major conformational change in the derivative relative to calmodulin. Interestingly, removal of the fluorenylmethyloxycarbonyl portion from the 1 : 1 adduct to give a “deprotected” 1 : 1 adduct resulted in apparent greater mobility of the probe, since the rotational correlation coefficient was found to be ∼1 nsec. Circular dichroic spectra were obtained over the wavelength interval 200–250 nm on the two adducts and on the deprotected 1 : 1 adduct. These derivatives, like calmodulin, exhibited a Ca2+-mediated increase in helicity, and the spectra of the adducts in the presence of a chelating agent and in the presence of saturating Ca2+ were similar to those obtained for calmodulin. Thus, the adducts have secondary structures similar to the native protein. Proton nuclear magnetic resonance spectra were determined in the aromatic region (∼6–8 ppm) for the deprotected 1 : 1 adduct before and after reduction of the nitroxide with ascorbate. The nitroxide had little effect on the chemical shifts of the two tyrosines and the single histidine relative to calmodulin, although the histidine C4 resonance was markedly altered by the addition of ascorbate. In order to explore in greater detail the tertiary structure of the 1 : 1 adduct, a reagent similar to I, but not paramagnetic, was synthesized. This compound II, α-N-(9′-fluorenylmethyloxycarbonyl)alanine N-hydroxysuccinimide ester, like I, forms a 1 : 1 adduct at Lys 75 and a 2 : 1 adduct at Lys 75 and Lys 148. Proton NMR spectra of adducts with II were not complicated by the relaxation effects arising from adducts with I; thus more definitive assignments could be made to the upfield resonances, including the fluorene protons. Again, it was possible to conclude that adduct formation had no major effect on the tertiary structure of the protein as monitored by chemical shifts associated with various residues. We conclude that modification of just Lys 75, a residue in the long connecting helix of calmodulin, does not lead to major changes in protein conformation but does interfere with the ability of calmodulin to stimulate an activatable form of bovine brain cyclic nucleotide phosphodiesterase.

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.

Functional properties of covalent .beta.-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation

The 31-residue neuropeptide porcine beta-endorphin was shown to inhibit the Ca2+-dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to beta-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular beta-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. We found that relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking beta-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked beta-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. In contrast, the 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 beta-endorphin 13-31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+-dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific acylation of calmodulin. Synthesis and adduct formation with a fluorenyl-based spin label.

The spin-labeling reagent, N4-(9'-fluorenylmethyloxycarbonyl)-4-amino-1-oxyl-4-succinimidyloxyca rbonyl- 2,2,6,6-tetramethylpiperidine, and the same enriched in 14C at the 4-formyl group, were synthesized as new acylating compounds for protein amino groups that can preserve charge. Porcine testicular calmodulin was modified with this reagent at pH 7.8 in the presence of Ca2+ under conditions that yielded a fairly homogeneous derivative as judged by electrophoretic analysis and tryptic digestion patterns. The tryptic peptides were separated by gel filtration and reverse-phase high-performance liquid chromatography, and the resulting, highly purified 14C-labeled peptides were hydrolyzed and their amino acid compositions determined. The results indicate that at least 87% of the modifications occur at lysyl residues 75 and 148, and the former appears to be the most reactive. This bilabeled calmodulin adduct does not activate a bovine brain cyclic nucleotide phosphodiesterase preparation. The fluorenylmethyloxycarbonyl portion of this inactive calmodulin derivative can, however, be removed by conditions that do not diminish native calmodulin activity in the phosphodiesterase assay. The resulting calmodulin adduct is active in the enzymic assay, although with diminished potency compared to calmodulin. The specificity of the reaction of this acylating reagent with calmodulin may be due to recognition of the tricyclic fluorene ring by the phenothiazine-binding sites since it was found that trifluoperazine inhibited the labeling reaction. Also, calmodulin was far more reactive to this reagent than were several other proteins. This is the first report of a specific, characterized lysine modification on calmodulin, and it is possible that other phenothiazine-binding proteins may also exhibit similar selectivity for acylation. Electron paramagnetic resonance spectra of the calmodulin adducts suggest a high degree of spin immobilization in both the Ca2+-free and Ca2+-saturated states.

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings ( Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca 2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy ( Renilla reniformis), mung bean seed ( Vigna radiata L. Wilczek), mushroom ( Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase ( K 0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K 0.5′s ranged from 19.6 ng/ml for bovine brain calmodulinto 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K 0.5′s for the activation of Ca 2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca 2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca 2+ (10% of maximum), but no effect in the absence of Ca 2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.

Structural and functional properties of calmodulin from the eukaryotic microorganism Dictyostelium discoideum.

Calmodulin was purified from the eukaryotic microorganism Dictyostelium discoideum and characterized in terms of its nearly complete primary structure and quantitative activator activity. The strategy for amino acid sequence analysis took advantage of the highly conserved structure of calmodulin and employed a new procedure for limited cleavage of calmodulin that uses a protease from mouse submaxillary gland. Fourteen amino acid sequence differences between Dictyostelium and bovine calmodulin were identified unequivocally, as well as an unmethylated lysine at residue 115 instead of N epsilon, N epsilon, N epsilon-trimethyllysine. Seven of the amino acid substitutions in Dictyostelium calmodulin are novel in that the residues at these positions are invariant in all calmodulin sequences previously examined, most notably an additional residue at the carboxy terminus. Comparison of the Dictyostelium calmodulin sequence with other calmodulin sequences shows that the region with the greatest extended sequence identity includes parts of the first and second structural domains and the interdomain region between domains 1 and 2. Dictyostelium calmodulin activated bovine brain cyclic nucleotide phosphodiesterase in a manner indistinguishable from that of bovine brain calmodulin. However, Dictyostelium calmodulin activated pea NAD kinase to a maximal level 4.6-fold greater than that produced by bovine brain calmodulin. This functional difference demonstrates the potential biological importance of the limited number of amino acid sequence differences between Dictyostelium calmodulin and other calmodulins and provides further insight into the structure, function, and evolution of the calmodulin family of proteins.

Preparation of an enzymatically active cross-linked complex between brain cyclic nucleotide phosphodiesterase and 3-(2-pyridyldithio)propionyl-substituted calmodulin.

Cyclic nucleotide phosphodiesterase (0.07 nM) was activated by near stoichiometric concentrations of [3-(2-pyridyldithio)propionyl]calmodulin (PDP-CaM) after initial incubation of these proteins at 200-fold higher concentrations; activity in assays with EGTA was 80% of that in the presence of Ca2+. The enzyme incubated with native calmodulin under identical conditions required approximately 1 nM for half-maximal activation, and no activation was observed in the absence of calcium. These data suggested formation of a covalent complex between phosphodiesterase and PDP-CaM. On high-performance gel-permeation chromatography in the presence of metal chelators, the complex appeared considerably larger than the native enzyme. Incubation of phosphodiesterase with the thiolated (inactivated) form of PDP-CaM did not change its chromatographic behavior, indicating that reactive sulfhydryl groups were involved in complex formation. Although the total activities recovered from chromatography were not significantly different, maximal activation of PDP-CaM-phosphodiesterase complex was only approximately 20%, whereas the control enzyme was activated 6-8-fold by Ca2+ plus calmodulin. Kinetics of cGMP hydrolysis in the presence of EGTA by the isolated complex differed from those of control enzyme but were indistinguishable from those of control enzyme assayed with saturating Ca2+ and CaM. The calmodulin antagonists W-7 and trifluoperazine had relatively little effect on activity of the PDP-CaM-phosphodiesterase complex. Incubation of the complex with dithiothreitol dramatically increased its Ca2+ and calmodulin responsiveness, suggesting that reduction of the disulfide cross-link released phosphodiesterase from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Assays for regulatory properties of polyamine-dependent protein kinase

Publisher Summary This chapter describes the methods for demonstrating calciumcalmodulin-dependent autophosphorylation of polyamine-dependent protein kinase. The assay for autophosphorylation of polyamine-dependent protein kinase requires the demonstration that the transfer of [ 32 P]HOPO 3 2- from [γ- 32 P]ATP to the protein kinase is markedly stimulated by calcium ion and calmodulin. The procedure involves the measurement of acid-stable [ 32 P]HOPO 3 2- incorporated into the protein kinase after the termination of the protein kinase reaction by a cold solution of ethylenediaminetetraacetic acid (EDTA) containing carrier bovine serum albumin. Calmodulin is assayed by its capacity to stimulate the hydrolysis of cyclic AMP to 5'-AMP catalyzed by bovine brain cyclic nucleotide phosphodiesterase. The polyamines spermidine and spermine inhibit the autophosphorylation reaction by polyamine-dependent protein kinase in the absence of calcium ion-calmodulin. Calcium ion-calmodulin complex reverses the inhibition caused by spermidine/spermine. This property can be demonstrated by comparing results from the assay mixtures. Representative data that demonstrate these regulatory effects on polyamine-dependent protein kinase are tabulated in the chapter.

Dictyostelium calmodulin: Affinity isolation and characterization

The Ca 2+-binding regulatory protein calmodulin (CaM) has been purified from the cellular slime mold, Dictyostelium discoideum. Isolation of homogeneous Dictyostelium CaM was accomplished in high yield by ion-exchange chromatography and Ca 2+-dependent affinity chromatography on phenothiazine-Sepharose 4B. This isolate has been demonstrated to possess the following physicochemical and functional properties characteristic of other CaM isolates: (i) a molecular weight ca. 16,000; (ii) an amino acid composition similar to other CaMs—with the notable exception that Dictyostelium CaM, as first determined by Bazari and Clarke [(1981) J. Biol. Chem. 256, 3598–3603] lacks the single trimethylated lysine (Tml) residue identified in nearly all CaMs purified to date; (iii) a CNBr peptide map similar to that of other CaMs; (iv) a Ca 2+-dependent shift in migration during native- and sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses; (v) ability to form Ca 2+-dependent complexes with rabbit skeletal muscle troponin I; and (vi) ability to activate in a Ca 2+-dependent manner bovine brain cyclic nucleotide phosphodiesterase.

Identification of beta-endorphin residues 14-25 as a region involved in the inhibition of calmodulin-stimulated phosphodiesterase activity.

The inhibition of the calmodulin-mediated stimulation of bovine brain cyclic nucleotide phosphodiesterase activity (3':5'-cyclic adenosine monophosphate 5'-nucleotidohydrolase, EC 3.1.4.17) by the 31-residue opiate peptide beta-endorphin has been investigated. Using conditions in which porcine brain calmodulin (6 nM) is limiting (i.e., to give a 3-fold, Ca2+-dependent stimulation of enzymic activity toward cyclic guanosine monophosphate), the domain of beta-endorphin responsible for the inhibition was mapped by using a series of deletion peptides. beta-Endorphin exhibited an ED50 of several micromolar under the conditions employed, and several amino-terminal deletion peptides were essentially as inhibitory as the parent peptide. Methionine enkephalin and various carboxy-terminal deletion peptides had no demonstrable effect at concentrations of 100-200 microM. Peptides 1-25 and 1-27 (C' fragment) inhibited the calmodulin-dependent activity of phosphodiesterase, but higher concentrations were required than of beta-endorphin. Studies using combined amino- and carboxy-terminal deletion peptides demonstrate that peptide 14-25 was the shortest peptide examined that was capable of inhibiting calmodulin stimulation of phosphodiesterase activity under the conditions used. There was no evidence to indicate that the amino-terminal region comprising residues 1-13 of beta-endorphin contributes to the measured inhibition of calmodulin-stimulated enzymic activity. The circular dichroic spectra of calmodulin, beta-endorphin, and mixtures of the two were obtained, and the ellipticity of the peptide-protein mixtures at 221 nm exceeded that expected by assuming simple additivity. This finding is consistent with a direct interaction of beta-endorphin with calmodulin which seems to lead to enhanced helicity of one or both components.(ABSTRACT TRUNCATED AT 250 WORDS)

African trypanosomes contain calmodulin which is distinct from host calmodulin

Studies were initiated to determine whether African trypanosomes utilize Ca2+ fluxes to coordinate complex morphological and biochemical life cycle changes. We have identified the ubiquitous intracellular Ca2+ receptor, calmodulin, in two developmental stages of Trypanosoma brucei rhodesiense. The transition from rapidly dividing, slender bloodstream trypomastigotes to slow growing procyclics in axenic culture was accompanied by changes in specific calmodulin content (3 μg/mg cell protein to 1 μg/mg cell protein, respectively) and a shift in intracellular calmodulin distribution, Trypanosome calmodulin is physically and functionally distinct from that of host tissues, including bovine brain and rat erythrocytes. It is similar to but distinct from Tetrahymena calmodulin. Comparisons among these proteins isolated from the four sources were made using the following criteria: (1) mobility on sodium dodecyl sulfate discontinuous polyacrylamide gels; (2) Ca2+-induced conformational changes; (3) CNBr-cleavage fragments; (4) activation of bovine brain cyclic nucleotide phosphodiesterase in both a Ca2+-dependent and calmodulin-dependent manner; (5) activation of human erythrocyte (Ca2+ + Mg2+)-ATPase; and (6) inhibition of calmodulin activity by trifluoperazine and penfluridol. Trifluoperazine but not trifluoperazine sulfoxide was cytotoxic to trypanosomes in vitro. Half maximal effect occurred at 15 μM. We conclude that calmodulin is a functional component of Africal trypanosomes and suggest that it plays an important role in mediating the host-parasite relationship.

A model for the regulation of the calmodulin-dependent enzymes erythrocyte Ca2+-transport ATPase and brain phosphodiesterase by activators and inhibitors.

Acidic phospholipids, unsaturated fatty acids and limited proteolysis mimic the activating effect of calmodulin on erythrocyte Ca2+-transport ATPase and on brain cyclic nucleotide phosphodiesterase, as has been reported previously in several studies. Three different antagonists of calmodulin-induced activation of these enzymes were tested for their inhibitory potency on the stimulation produced by the other activators. Trifluoperazine and penfluridol were found to antagonize all the above mentioned types of activation of Ca2+-transport ATPase in the same concentration range. Both inhibitors also can reverse the activation of phosphodiesterase by oleic acid, phosphatidylserine and calmodulin at similar concentrations. However, in contrast with erythrocyte Ca2+-transport ATPase, activation of phosphodiesterase by limited tryptic digestion cannot be antagonized by penfluridol and trifluoperazine. Calmidazolium, formerly referred to as compound R 24571, was found to be a relatively specific inhibitor of calmodulin-induced activation of phosphodiesterase and Ca2+-transport ATPase, since antagonism of the other activators required much higher concentrations of the drug. The results suggest that the investigated drugs exert their inhibitory effect on calmodulin-regulated enzymes not solely via their binding to calmodulin but may also interfere directly with the calmodulin effector enzyme. In addition, a general mechanism of activation and inhibition of calmodulin-dependent enzymes is derived from our results.

Calmodulin Purification and Quantitation from Bovine Mammary Tissue

Calmodulin, a multifunctional calcium binding protein, has been purified to apparent homogeneity from bovine lactating mammary tissue. Calmodulin was purified by heat treatment and sequential anion exchange and gel exclusion chromatographies. The molecular weight of the purified protein was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18,000 daltons. The purified protein produced a 10-fold stimulation of the activity of partially purified bovine brain cyclic nucleotide phosphodiesterase in a calcium-dependent manner. This report details a method for the assay of biologically active calmodulin from lactating mammary tissue based on its stimulation of partially purified bovine brain phosphodiesterase. We report concentrations (μg/g tissue, mg protein, mg deoxyribonucleic acid) of biologically active calmodulin in bovine mammary tissue.

Inhibition of bovine brain cyclic nucleotide phosphodiesterase by a proteinaceous factor from Escherichia coli.

An inhibitory factor for Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase of bovine brain was present in the soluble fraction of Escherichia coli. The factor was heat-stable but trypsin sensitive. The activity of brain phosphodiesterase supported by Ca2+ and calmodulin was inhibited by the factor in a dose dependent manner, but the basal activity was not affected. The inhibition of phosphodiesterase induced by the factor could be abolished by adding large amount of calmodulin, but not by increasing concentration of Ca2+. It was suggested that the factor interacted with calmodulin and thereby inhibited the phosphodiesterase. The factor may be a calmodulin-binding protein in E. coli.

Stimulation of cyclic nucleotide phosphodiesterase by products of phosphatidylinositol metabolism catalyzed by phospholipase A 2

Products of phospholipase A 2-catalyzed hydrolysis of phosphatidylinositol, lysophosphatidylinositol, and unsaturated fatty acid are very effective stimulators of a partially purified bovine brain cyclic nucleotide phosphodiesterase. More than 10-fold of calcium-independent stimulation can be achived by lysophatidylinositol or oleic acid. The degree of stimulation is comparable to that by calcium-mediated calmodulin. The other lysophospholipid which shows comparable stimulation is lysophosphatidylserine although at a higher concentration. Diacylphosphoglycerides are inactive. Kinetic studies showed that lysophosphatidylinositol, like calmodulin, increased the V max without affecting the apparent K m for adenosine 3′,5′-cyclic phosphate (cAMP).