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PubMed1985

[Effect of neurotropic drugs on calmodulin and troponin C-dependent processes].

The effects of neurotropic compounds on Ca-binding proteins (calmodulin, troponin C) were investigated. It was shown that the majority of neuroleptics of the phenothiazine group effectively interact with the both proteins and inhibit calmodulin-dependent cyclic nucleotide phosphodiesterase and Ca2+-activated actomyosin. ATPase. Neuroleptics of the butyrophenone group as well as imipramine and diphenehydramine having a low efficiency interact only with calmodulin. Methophenazine, a phenothiazine neuroleptic, being an effective inhibitor of calmodulin and of calmodulin-dependent phosphodiesterase, does not influence troponin C or Ca-dependent actomyosin ATPase. Therefore, this compound may be used as a convenient tool in the study of processes controlled by these Ca-binding proteins. It is concluded that troponin C possesses Ca-dependent sites which bind pharmacological agents structurally similar to that of calmodulin. However, these sites bind pharmacological agents with a low efficiency and exhibit selectivity towards certain drugs. Despite the obvious homology of the both Ca-binding proteins, i.e., calmodulin, troponin C, their effects on the processes under their control appear to be selective.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1988

Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum.

Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of either erythrocyte membrane Ca2+-ATPase activity or cardiac sarcoplasmic reticulum phosphorylation are very similar. Affinity labeling of troponin subunits with 125I- and benzophenone-4-maleimide-labeled CaM demonstrates CaM binding to troponin I (TnI) and troponin T (TnT) in binary complexes, as well as to both subunits in the CaM.TnI.TnT ternary complex. This suggests that both subunits are within 10 A of Cys27 of calmodulin. Affinity labeling of cardiac sarcoplasmic reticulum vesicles with 125I- and benzophenone-4-maleimide-labeled CaM exhibits a Ca2+- and Mg2+-dependent labeling of phospholamban, as shown previously with bovine calmodulin (Louis, C.F., and Jarvis, B. (1982) J. Biol. Chem. 257, 15187-15191). Thus, it appears that Ca2+-binding site I of calmodulin is at or near binding sites of calmodulin for TnI, TnT, and phospholamban. Analysis of the time-resolved fluorescence decay curves of I-EDANS-labeled calmodulin indicates a major component with a lifetime of 11.9 ns (+Ca2+), which accounts for 81% of the total fluorescence. The lifetime decreases slightly to 11.3 ns in the absence of Ca2+. Fluorescence anisotropy experiments indicate that I-EDANS-labeled CaM binds TnI with Kd = 6 x 10(-8) M in the presence of Ca2+. This study suggests that these single-site derivatives will be useful for characterizing a variety of calmodulin-receptor interactions because they lack ambiguities inherent in less specific labeling methods.

Animals↗

PubMed1987

Fast activation of Ca2+-ATPases in plasma membranes from cardiac muscle and from ascites carcinoma cells: a possible function of endogenous calmodulin.

Content of endogenous calmodulin, binding of calmodulin to, and Ca2+-ATPase activity in plasma membranes of cardiac muscle. Ehrlich ascites carcinoma (EAC) cells and erythrocytes were examined. The content of endogenous calmodulin in cardiac and EAC cells was shown to be considerably higher than in erythrocyte membranes. Ca2+-independent binding of calmodulin to cardiac and EAC cell membranes was found to be realized by some low molecular weight proteins. Ca2+-ATPases in cardiac and EAC cell membranes differ from those in erythrocytes with respect to their activation by Ca2+ and calmodulin. The erythrocyte enzyme is strongly stimulated by exogenous calmodulin and reaches its maximum activity about 2 min after Ca2+-addition. In contrast, the Ca2+-ATPases in cardiac and EAC cell plasma membranes cannot be considerably stimulated by exogenous calmodulin and are instantaneously activated by Ca2+.

Animals↗

PubMed1985

P-57 is a neural specific calmodulin-binding protein.

P-57 is a novel calmodulin-binding protein which has recently been isolated from bovine cerebral cortex (Andreasen, T. J., Luetje, C. W., Heideman, W., and Storm, D. R. (1983) Biochemistry 22, 4615-4618). In contrast to all other calmodulin-binding proteins characterized thus far, P-57 has equivalent or higher affinity for calmodulin in the absence of free Ca2+ compared to the presence of Ca2+. In this study, the distribution of P-57 in other tissues and within brain was examined using a radioimmune assay and photoaffinity labeling with azido-125I-calmodulin. P-57 was not found in tissues other than brain, retina, and spinal cord. Within brain, P-57 levels varied from 0.1% of the total protein in white matter regions to about 0.5% in cell body-rich fractions. The protein was found in both membrane and soluble fractions. P-57 is the most abundant calmodulin-binding protein in brain and appears to be neural specific. The concentrations of P-57 in brain and its affinity for calmodulin in the absence of Ca2+ are sufficient to complex a significant fraction of the total calmodulin present.

Animals↗

PubMedDate not supplied

[Ca2+-calmodulin-activated cyclic nucleotide phosphodiesterase from the rabbit myometrium].

Two forms of soluble phosphodiesterase of cyclic nucleotides separating by DEAE-cellulose ion-exchange chromatography and not only differing in physicochemical and catalytic parameters but also differently regulated by calmodulin are found in the doe myometrium. Calmodulin with 10(-7)-10(-5) M concentrations of Ca2+ promotes the two-fold activation of the 3':5'-AMP (but not of 3':5'-GMP) hydrolysis by the first form of phosphodiesterase. Trifluoperazine (10 microM) lowers the activating action of calmodulin. The second form of soluble phosphodiesterase is not sensitive to the action of both calmodulin and Ca2+. 3':5'-GMP (10 microM) inhibits the 3':5'-AMP hydrolysis by the first form of phosphodiesterase; calmodulin exerts no effect on this process. The data obtained testify to the possible participation of Ca2+ and calmodulin in Ca2+-calmodulin-dependent phosphodiesterase regulation of the content of cyclic nucleotides (3':5'-AMP, in particular) in the doe myometrium.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1987

A novel mechanism for the inhibition of adenylate cyclase via inhibitory GTP-binding proteins. Calmodulin-dependent inhibition of the cyclase catalyst by the beta gamma-subunits of GTP-binding proteins.

The adenylate cyclase catalytic protein partially purified from rat brain membranes was activated by the stimulatory GTP-binding protein (Gs), forskolin, and Ca2+-calmodulin. The Ca2+-calmodulin-stimulated activity was markedly, but the Gs- or forskolin-stimulated activity was essentially not, inhibited by low concentrations of the beta gamma-subunits of the inhibitory GTP-binding protein (Gi). The inhibition appeared to be competitive with calmodulin. On the other hand, the association of increasing amounts of beta gamma with the alpha of Gi, which was measured based on the ADP-ribosylation by islet-activating protein, pertussis toxin, was apparently competed by Ca2+-calmodulin. Furthermore, beta gamma bound to calmodulin-Sepharose in the presence of Ca2+, but not in its absence. Thus, the direct interaction of beta gamma with calmodulin is a likely mechanism involved in beta gamma-induced inhibition of the calmodulin-stimulated adenylate cyclase.

Adenylate Cyclase Toxin↗

PubMed1987

Partial repair of deamidation-damaged calmodulin by protein carboxyl methyltransferase.

Modification of calmodulin by protein carboxyl methyltransferase requires deamidation of one or more labile asparagine residues (Johnson, B.A., Freitag, N. E., and Aswad, D. W. (1985) J. Biol. Chem. 260, 10913-10916). We now show that deamidation results in the generation of two altered forms of calmodulin, designated A and B, which can be separated by electrophoresis under nondenaturing conditions. The A form is characterized by a larger apparent molecular radius, has only 10% the activity of native calmodulin when assayed for its ability to activate a Ca2+/calmodulin-dependent protein kinase from rat brain, and serves as an excellent substrate for the methyltransferase. The B form more closely resembles native calmodulin: it has an apparent molecular radius more like the native, exhibits about 40% the activity of native calmodulin, and is a relatively poor methyl acceptor. Evidence suggests that the A and B forms probably contain isoaspartate (A) and aspartate (B) in place of Asn-60 and/or Asn-97. Incubation of the A form with methyltransferase and S-adenosyl-L-methionine converts about half of the A form to an electrophoretic band indistinguishable from the B form. The activity of this partly converted calmodulin rises to 30-50% that of native calmodulin. These observations imply that the methyltransferase may have a biological role in restoring activity to proteins which contain abnormal isoaspartyl peptide bonds resulting from asparagine deamidation.

Animals↗

PubMed1987

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence.

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.

Amino Acid Sequence↗

PubMed1987

Microcalorimetric investigation of the interactions in the ternary complex calmodulin-calcium-melittin.

Flow microcalorimetric titrations of calmodulin with melittin at 25 degrees C revealed that the formation of the high-affinity one-to-one complex in the presence of Ca2+ (Comte, M., Maulet, Y., and Cox, J. A. (1983) Biochem, J. 209, 269-272) is entirely entropy driven (delta H0 = 30.3 kJ X mol-1; delta S0 = 275 J X K-1 X mol-1). Neither the proton nor the Mg2+ concentrations have any significant effect on the strength of the complex. In the absence of Ca2+, a nonspecific calmodulin-(melittin)n complex is formed; the latter is predominantly entropy driven, accompanied by a significant uptake of protons and fully antagonized by Mg2+. Enthalpy titrations of metal-free calmodulin with Ca2+ in the presence of an equimolar amount of melittin were carried out at pH 7.0 in two buffers of different protonation enthalpy. The enthalpy and proton release profiles indicate that: protons, absorbed by the nonspecific calmodulin-melittin complex, are released upon binding of the first Ca2+; Ca2+ binding to the high-affinity configuration of the calmodulin-melittin complex displays an affinity constant greater than or equal to 10(7) M-1, i.e. 2 orders of magnitude higher than that of free calmodulin; the latter is even more entropy driven (delta H0 = 7.2 kJ X site-1; delta S0 = 158 J X K-1 X site-1) than binding to free calmodulin (delta H0 = 4.7 kJ X site-1; delta S0 = 112 J X K-1 X site-1), thus underlining the importance of hydrophobic forces in the free energy coupling involved in the ternary complex.

Animals↗

PubMed1985

Binding of calmodulin to the microfilament network correlates with induction of a macrophage tumoricidal response.

Induction of mouse peritoneal macrophage cytotoxicity against SV3T3, a line of virally transformed mouse cells correlated with the distribution of cytoplasmic calmodulin in the macrophages. The organization of the cytoskeleton was examined by fluorescent microscopy and by transmission electron microscopy, using immunogold tagging after Triton-X-100 (TX-100) extraction of the macrophages. Macrophages that had been activated to a tumoricidal state in vivo by vaccinia virus or in vitro by lymphokine stimulation displayed cytoskeletal networks that were more extended and weblike than did resident macrophages. The organization of microfilaments and microtubules in the cytoskeleton was displayed by using either anti-actin or anti-tubulin. Immunogold labeling of tumoricidal macrophage cytoskeletons with anti-calmodulin revealed strong binding to the microfilament network and no binding to microtubules. Anti-calmodulin reacted weakly with the cytoskeletal network of resident macrophages, and this was not demonstrably greater than the reaction with normal sheep serum. However, resident macrophages displayed a high density of calmodulin (CAM) associated with unidentifiable structures in the perinuclear region when reacted with anti-calmodulin. These characteristic distributions of CAM in resident and activated macrophages was confirmed by immunofluorescence. The total and cytoskeletal-associated amounts of calmodulin per unit of protein were determined by radioimmune assay and 125I labeling followed by SDS-PAGE. No statistically significant differences were detected between resident and activated macrophages in either the total cell or cytoskeleton fractions. In summary, our results suggest that induction of tumoricidal activity of mouse peritoneal macrophages correlates with the translocation of calmodulin to the microfilament network of the cytoskeleton.

Animals↗

PubMed1985

[Mechanism of involvement of calmodulin in the regulation of affinity to Ca2+ and maximum activity of Ca-pump in the erythrocyte membrane].

The activity of the Ca-pump in inside-out oriented human erythrocyte membrane vesicles was studied with the use of 45Ca and membrane filters. It was found that trifluoroperazine fully inhibits the calmodulin-induced increase in the maximal activity of the Ca-pump without affecting the calmodulin-stimulated increase in the Ca-pump affinity for Ca2+. The dependence of calcium concentrations of calmodulin-stimulated components of the Ca-pump activity, both inhibited and noninhibited by trifluoroperazine, as well as the dependence on calcium concentrations of the fluorescence intensity of N-phenyl-1-naphthylamine were analyzed. This analysis revealed essential differences between the mechanisms of calmodulin action on the maximal activity of the Ca-pump and its affinity for Ca2+. The maximal activity was elevated by addition of the Ca-calmodulin complex, whereas the increase in the affinity for Ca2+ was induced by calmodulin alone. These findings were supported by data on the dependence of the Ca-pump activity on calmodulin concentrations at low and saturating concentrations of Ca2+ as well as by data obtained in the study on moderate treatment of erythrocyte membranes with trypsin.

Biological Transport↗

PubMed1984

Interaction of quaternary phenothiazine salts with calmodulin.

To clarify the role of electrostatic forces in the binding of phenothiazines to calmodulin, the quaternary methiodide salts of several 10-alkylaminophenothiazines were prepared and examined for anticalmodulin activity. In addition, the methiodide salt of [3H]chlorpromazine was prepared and its binding to calmodulin was studied. Both the hydrochloride and methiodide salts of a variety of 10-alkylaminophenothiazines inhibited the calmodulin-induced activation of phosphodiesterase at concentrations that had no effect on the basal activity of the enzyme. The IC50 values for the inhibition of calmodulin by the methiodide salts were similar to those of the corresponding hydrochloride salts, with the compounds displaying the order of potency: trifluopromazine greater than chlorpromazine greater than promazine greater than promethazine greater than chlorpromazine sulfoxide. The acidic compound 3-(2-chloro-10-phenothiazinyl)propionic acid displayed no anticalmodulin activity at concentrations up to 80 microM; at higher concentrations the compound inhibited both basal and calmodulin-stimulated enzyme activity. Other results showed that both chlorpromazine hydrochloride and chlorpromazine methiodide bound to calmodulin in a Ca++-dependent manner. However, the binding of chlorpromazine hydrochloride decreased markedly at alkaline pHs, whereas the binding of chlorpromazine methiodide was not pH-dependent. The fact that the binding of the nondissociable methiodide salt is not pH-dependent, whereas the binding of the hydrochloride salt decreases at pHs at which the drug would be nonionized, indicates that it is the cationic species of the phenothiazine that binds to calmodulin and inhibits its activity.

Animals↗

PubMed1982

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum.

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.

Adenosine Triphosphatases↗

PubMed1984

Localization of calmodulin in differentiating pulmonary type II epithelial cells.

Pulmonary surfactant is synthesized by alveolar type II pneumonocytes and stored in inclusions called lamellar bodies. In the present study we have investigated the role of the calcium-binding protein, calmodulin, in regulating surfactant secretion in differentiating rat fetal type II pneumonocytes. Lamellar body secretion is stimulated in differentiating type II cells in vitro by the calcium ionophore, A23187. A23187-induced secretion is blocked by the phenothiazine drugs, trifluoperazine and chlorpromazine, but is unaffected by the inactive analogs, trifluoperazine sulfoxide and chlorpromazine sulfoxide. Immunofluorescence studies on cultured type II pneumonocytes show that the percentage of Nomarski-dense intracellular granules, which stain positively with anticalmodulin antibody, increases when the cells are stimulated with the calcium ionophore, A23187. Since these Nomarski-dense granules are positively stained by phosphine-3R, these results indicate that increased amounts of immunoreactive calmodulin appear associated with lamellar body surfaces when the cells are stimulated for secretion. In addition, ultrastructural localization of calmodulin on isolated lamellar bodies using protein A-colloidal gold indicates that calmodulin is present on their outer surfaces. The endogenous calmodulin present on the isolated lamellar bodies is not removed by treatment of the lamellar bodies with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Using the same localization technique, we found that isolated lamellar bodies are capable of binding exogenously added calmodulin in the presence of calcium. Taken together, these results implicate calmodulin in pulmonary surfactant secretion.

Animals↗

PubMed1983

Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation.

Cardiac sarcoplasmic reticulum contains an endogenous calcium-calmodulin-dependent protein kinase and a 22,000-Da substrate, phospholamban. This kinase is half-maximally activated (EC50) by 3.8 +/- 0.3 microM calcium and is absolutely dependent on exogenous calmodulin (EC50 = 49 nM). To determine the effect of this phosphorylation on calcium transport, sarcoplasmic reticulum vesicles (0.5 mg/ml) were preincubated under conditions for optimal phosphorylation (50 mM potassium phosphate, pH 7.0, 10 mM MgCl2, 0.5 mM EGTA, 0.478 mM CACl2, 0.1 microM calmodulin, 0.5 mM ATP). Control sarcoplasmic reticulum was preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both control and phosphorylated vesicles were centrifuged and resuspended in 0.3 M sucrose, 20 mM Tris-HCl, 100 mM KCl, pH 7.0, to remove calmodulin and subsequently assayed for calcium (45Ca) transport in the presence of 2.5 mM Tris-oxalate. Phosphorylation of sarcoplasmic reticulum vesicles by calcium-calmodulin-dependent protein kinase resulted in a significant increase (2- to 4-fold) in the rate of calcium transport at low calcium concentrations (less than 3 microM), while calcium transport was minimally affected at higher calcium. Hill coefficients (n) derived from Hill plots of transport data showed no difference between control and phosphorylated sarcoplasmic reticulum (n = 2.0), indicating that phosphorylation does not alter the cooperativity between calcium sites on the calcium pump. The EC50 for calcium activation of calcium transport by control vesicles was 0.86 +/- 0.1 microM calcium, and phosphorylation of phospholamban decreased this value to 0.61 +/- 0.07 microM calcium (n = 7, p less than 0.028), indicating an increase in the apparent affinity for calcium upon phosphorylation. These results were found to be specific for calcium-calmodulin-dependent phosphorylation of phospholamban. Control experiments on the effects of the reactants used in the phosphorylation assay and subsequent centrifugation of sarcoplasmic reticulum showed no alteration of the rate of calcium transport. Therefore, the calcium pump in cardiac sarcoplasmic reticulum appears to be regulated by an endogenous calcium-calmodulin-dependent protein kinase, and this may provide an important regulatory mechanism for the myocardium.

Adenosine Triphosphatases↗

PubMed1984

Stimulation of the purified erythrocyte Ca2+-ATPase by tryptic fragments of calmodulin.

Highly purified tryptic peptides of calmodulin have been obtained by high-performance liquid chromatography. Tryptic cleavage of calmodulin in the presence of Ca2+ results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-148. In the absence of Ca2+, trypsin cleavage yields fragments 1-106, 1-90, and 107-148. Only fragments 78-148 and 1-106 are still able to stimulate the purified Ca2+-ATPase of erythrocytes, albeit much less efficiently on a molar basis, than intact calmodulin. On the other hand, the same fragments were unable to stimulate the calmodulin-dependent cyclic nucleotide phosphodiesterase, even at 1000-fold molar excess (shown also by Newton, D.L., Oldewurtel, M.D., Krinks, M.H., Shiloach, J., and Klee, C.B. (1984) J. Biol. Chem. 259, 4419-4426). This points to the importance of the carboxyl-terminal half of calmodulin and especially of Ca2+-binding region III in the interaction of calmodulin with the Ca2+-ATPase and provides clear evidence that calmodulin interacts differently with different targets. Oxidation of methionine(s) of fragment 78-148 with N-chlorosuccinimide removes the ability of this fragment to stimulate the ATPase.

Amino Acids↗

PubMed1980

Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase.

Calmodulin-dependent cyclic nucleotide phosphodiesterase was purified from bovine brain to apparent homogeneity by a new procedure involving DEAE-cellulose, Affi-Gel blue, calmodulin-Sepharose 4B, and Sephadex G-200 column chromatographies. The enzyme was purified more than 3,000-fold from the brain extracts with greater than 12% yield. The purified phosphodiesterase could be activated 10- to 15-fold by calmodulin and Ca2+ to a specific enzyme activity of more than 300 mumol of cAMP hydrolyzed/min/mg of protein. Molecular weight of the enzyme was determined to be 115,800 by the sedimentation equilibirum method or 124,000 from the sedimentation constant and Stokes radius of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme showed a single protein band with an apparent molecular weight of 58,000. These results suggested that the calmodulin-dependent phosphodiesterase from bovine brain has a subunit structure of alpha2. Molecular weight of the complex of calmodulin and phosphodiesterase was the complex of calmodulin and phosphodiesterase was also calculated from the sedimentation constant and Stokes radius to be 159,000. Since calmodulin has a molecular weight of about 17,000, the result indicated that the stoichiometry of the complex is calmodulin2 alpha2. The catalytic subunit of cylic AMP-dependent protein kinase was found to catalyze the phosphorylation of the purified phosphodiesterase with the incorporation of 2 mol of phosphate/mol of the enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1981

[Effect of pH on Ca-binding properties of calmodulin and on its interaction with Ca-dependent phosphodiesterase of cyclic nucleotides].

The fluorescence of dansyl immobilized on bovine brain calmodulin is sensitive to Ca2+. This effect is due to Ca2+ attachment to specific Ca2+-binding sites of calmodulin and is maintained within a wide range of pH. The native and dansyl-modified calmodulin preparations exert similar activating effects on Ca-dependent phosphodiesterase of cyclic nucleotides and have practically the same affinity for the enzyme. Using fluorescence measurements of the calmodulin--dansyl conjugate, it was shown that the decrease of pH from 9.0 down to 6.0 gradually decreases the constant of Ca2+ binding to calmodulin from 1.5 . 10(10) M-1 to 1.6 . 10(6) M-1. This decrease of pH does not affect the calmodulin affinity for phosphodiesterase. The activating effect of calmodulin on phosphodiesterase is more pronounced at acidic pH values (6.0-7.0) than at alkaline pH values (8.0-9.0).

3',5'-Cyclic-AMP Phosphodiesterases↗