Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.1 (phosphodiesterase)
18,767 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 3-A crystal structure of calmodulin indicates that it has a polarized tertiary arrangement in which calcium binding domains I and II are separated from domains III and IV by a long central helix consisting of residues 65-92. To investigate the functional significance of the central helix, mutated calmodulins were engineered with alterations in this region. Using oligonucleotide-primed site-directed mutagenesis, Thr-79 was converted to Pro-79 to generate CaMPM. CaMPM was further mutated by insertion of Pro-Ser-Thr-Asp between Asp-78 and Pro-79 to yield CaMIM. Calmodulin, CaMPM, and CaMIM were indistinguishable in their ability to activate calcineurin and Ca2+-ATPase. All mutated calmodulins would also maximally activate cGMP-phosphodiesterase and myosin light chain kinase, however, the concentrations of CaMPM and CaMIM necessary for half-maximal activation (Kact) were 2- and 9-fold greater, respectively, than CaM23. Conversion of the 2 Pro residues in CaMIM to amino acids that predict retention of helical secondary structure did not restore normal calmodulin activity. To investigate the nature of the interaction between mutated calmodulins and target enzymes, synthetic peptides modeled after the calmodulin binding region of smooth and skeletal muscle myosin light chain kinase were prepared and used as inhibitors of calmodulin-dependent cGMP-phosphodiesterase. The data suggest that the different kinetics of activation of myosin light chain kinase by CaM23 and CaMIM are not due to differences in the ability of the activators to bind to the calmodulin binding site of this enzyme. These observations are consistent with a model in which the length but not composition of the central helix is more important for the activation of certain enzymes. The data also support the hypothesis that calmodulin contains multiple sites for protein-protein interaction that are differentially recognized by its multiple target proteins.
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PMID:Functional significance of the central helix in calmodulin. 284 23

The effect of four slow Ca2+ channel blockers (felodipine, nifedipine, prenylamine and bepridil) that possess the ability to bind to calmodulin (CaM) section and to inhibit myosin light chain kinase (MLCK) on CaM-regulated Ca2+ pumping ATPase of cardiac sarcolemma (SL) and brain cyclic AMP phosphodiesterase (PDE) was studied. The ability of these drugs to inhibit Ca2+ pumping ATPase correlated with their inhibitory effect on CaM-activated Ca2+-dependent PDE. Nifedipine was unable to inhibit markedly both enzymes. Prenylamine also was a weak inhibitor, which was unexpected because of its CaM binding potency. Felodipine (10-50 microM) and bepridil (50 microM) markedly reduced activities of SL Ca2+ pumping ATPase and PDE. Striking differences were, however, demonstrated when Ca2+ and CaM concentrations, respectively, were increased. Previously it was reported that inhibition of the SL Ca2+ pumping ATPase by the CaM antagonist calmidazolium could be overcome by increasing Ca2+ concentrations (J. M. J. Lamers and J. T. Stinis, Cell Calcium 4, 281-294, 1983). Felodipine (10-50 microM) in the present study, appeared to be equipotent with calmidazolium in reducing Ca2+ pumping ATPase, but increasing Ca2+ up to 12.2 microM could not counteract this effect. Felodipine (2-10 microM) also inhibited brain PDE noncompetitively with respect to CaM contrary to the competitive effectors calmidazolium and bepridil. On the other hand, bepridil (10-20 microM) decreased or increased Ca2+ pumping ATPase activity depending on the Ca2+ concentration (0.29 and 12.2 microM, respectively) used. These findings suggest at least two types of CaM antagonists, which can be discriminated on basis of their inhibition patterns of PDE and heart SL Ca2+ pumping ATPase.
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PMID:Slow calcium channel blockers and calmodulin. Effect of felodipine, nifedipine, prenylamine and bepridil on cardiac sarcolemmal calcium pumping ATPase. 293 41

The cytosol fraction of an extract of Xenopus laevis ovaries contains a protein inhibitor that can specifically block the activation of calmodulin-sensitive cyclic nucleotide phosphodiesterase (PDE I) found in that tissue. This inhibitor was purified by DEAE-cellulose chromatography, gel filtration on Sephacryl S-200, and affinity chromatography on calmodulin-Sepharose. It has a molecular weight of approximately 90,000, and is heat-labile and susceptible to inactivation by chymotrypsin. The inhibitor blocks calmodulin activation of cyclic nucleotide phosphodiesterases from amphibian ovary and bovine brain and of the myosin light chain kinase from rabbit smooth muscle, but does not affect the activity of a calmodulin-insensitive cyclic nucleotide phosphodiesterase. The inhibitor not only affects the activation of Xenopus PDE I and of the bovine brain phosphodiesterase by calmodulin, but also inhibits the stimulation of these enzymes by lysophosphatidylcholine. The inhibitor also acts on PDE I activated by partial tryptic proteolysis, but the enzyme fully activated by trypsin is only slightly susceptible to inhibition by this protein. The inhibition of PDE I activation caused by this ovarian factor can be reversed by adding excess amounts of calmodulin or lysophosphatidylcholine. The presence of this inhibitor provides a possible explanation for the previously observed inactivity of PDE I in vivo.
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PMID:A protein inhibitor of calmodulin-regulated cyclic nucleotide phosphodiesterase in amphibian ovaries. 299 90

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.
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PMID:Ca2+-dependent cyclic nucleotide phosphodiesterase is activated by poly(L-aspartic acid). 300 Apr 29

The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.
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PMID:Calcium binding to complexes of calmodulin and calmodulin binding proteins. 300 73

Alteration of residues 82-84 in the alpha-helix that links the two halves of calmodulin results in a differential effect on activator activity. Previous studies (Lukas, T. J., Burgess, W. H., Prendergast, F. G., Lau, W., and Watterson, D. M. (1986) Biochemistry 25, 1458-1464) indicated the importance of positive charge clusters in the calmodulin-binding protein, myosin light chain kinase. This suggested the possible importance of complementary negative charge clusters in calmodulin. By using an efficient cassette mutagenesis approach and a synthetic calmodulin gene (Roberts, D. M., Crea, R., Malecha, M., Alvarado-Urbina, G., Chiarello, R. H., and Watterson, D. M. (1985) Biochemistry 24, 5090-5098), this possibility was directly addressed by engineering a new calmodulin, VU-8 calmodulin, in which the glutamate cluster at residues 82-84 in the synthetic gene product (VU-1 calmodulin) was replaced by three lysines. VU-8 calmodulin activated phosphodiesterase to the same maximal extent as VU-1 calmodulin, although there was an alteration in the concentration of calmodulin required for half-maximal stimulation. In contrast, myosin light chain kinase was activated to only 30% of maximal activity and NAD kinase was not activated. These results provide insight into the functional role of the unusual central helix structure found in the calmodulin family of proteins and indicate that different, although possibly overlapping, chemical complementarities are employed in the interaction between calmodulin and its various physiological targets.
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PMID:Site-specific mutagenesis of the alpha-helices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin. 302 8

High-affinity antibodies against calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase and protein phosphatase (calcineurin) were purified and characterized. Rabbit anti-phosphodiesterase antibody did not react with other phosphodiesterases or with the regulatory subunits of cAMP-dependent protein kinase. Affinity-purified goat anti-calcineurin antibody recognized both the 61-kDa catalytic subunit and the 18-kDa Ca2+-binding subunit of the phosphatase. Neither antibody reacted with CaM, several CaM-binding proteins (calmodulin-dependent protein kinase, myosin light chain kinase, fodrin), or other cytosolic proteins from brain. The antibodies were used to compare the cellular localization of these two CaM-dependent enzymes in rat brain. Both calcineurin and phosphodiesterase were found predominantly in nerve cells; however, phosphodiesterase was restricted to very specific neuronal populations. Phosphodiesterase was prominent in the somatic cytoplasm and dendrites of regional output neurons--e.g., cerebellar Purkinje cells and hippocampal and cortical pyramidal cells. The extensive and uniform staining in the dendrites was consistent with postsynaptic localization and suggested an important function for this enzyme in neurons that integrate multiple convergent inputs. Calcineurin was present in virtually all classes of neurons, with immunoreactivity confined primarily to cell bodies. Both diffuse cytoplasmic staining and characteristic punctate staining of cell bodies were observed; the latter suggested compartmentalization of calcineurin at or near the plasma membrane. The results of this study demonstrate that calcineurin and phosphodiesterase are differentially localized in the central nervous system. Thus, the expression and compartmentalization of CaM-binding proteins may be highly regulated and specific for particular differentiated nerve cell types.
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PMID:Differential localization of calmodulin-dependent enzymes in rat brain: evidence for selective expression of cyclic nucleotide phosphodiesterase in specific neurons. 302 62

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca2+ are mediated by the Ca2+-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca2+-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca2+-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an 125I-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Ca2+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100 degrees C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased 125I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52,000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membrane. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggest a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.
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PMID:Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes. 358 33

We have prepared and partially characterized a lissamine-rhodamine B fluorescent analogue of calmodulin, LRB-CM. The analogue had a dye/protein ratio of approximately 1.0 and contained no free dye or contaminating labeled proteins. LRB-CM was indistinguishable from native calmodulin upon SDS PAGE and in assays of phosphodiesterase and myosin light chain kinase. The emission spectrum of LRB-CM was insensitive to changes in pH, ionic strength, and temperature over the physiological range, but the apparent quantum yield was influenced somewhat by divalent cation concentration. LRB-CM injected into living Swiss 3T3 fibroblasts became associated with nitrobenzoxadiazole-phallacidin staining stress fibers in some interphase cells. LRB-CM and acetamidofluorescein-labeled actin co-injected into the same cell both became associated with fibers in some cells, but in most cases association of the two analogues with fibers was mutually exclusive. This suggests that calmodulin may differ from actin in the timing of incorporation into stress fibers or that we have distinguished distinct populations of stress fibers. We were able to detect no direct interaction of LRB-CM with actin by fluorescence photobleaching recovery (FRAP) of aqueous solutions. Interaction of LRB-CM with myosin light chain kinase also was not detected by FRAP. This suggests that the mean lifetime of the calmodulin-myosin light chain kinase complex is too short to affect the diffusion coefficient of calmodulin. We examined various fluorescent derivatives of proteins and dextrans as suitable control molecules for quantitative fluorescent analogue cytochemistry in living cells. Fluorescein isothiocyanate-dextrans were found to be preferable to all the proteins tested, since their mobilities in cytoplasm were inversely dependent on molecular size and there was no evidence of binding to intracellular components. In contrast, FRAP of LRB-CM in the cytoplasm of living 3T3 cells suggested that the analogue interacts with intracellular components with a range of affinities. The mobility of LRB-CM in the cytoplasm was sensitive to treatment of the cells with trifluoperazine, which suggests that at least some of the intracellular binding sites are specific for calmodulin in the calcium-bound form. FRAP of LRB-CM in the nuclei of living 3T3 cells indicated that the analogue was highly mobile within the nucleus but entered the nucleus from the cytoplasm much more slowly than fluorescein isothiocyanate-dextran of comparable molecular size and much more slowly than predicted from its mobility in cytoplasm.
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PMID:Behavior of a fluorescent analogue of calmodulin in living 3T3 cells. 404 38

A photoaffinity label for calmodulin-binding proteins was prepared from 125I-labeled calmodulin (125I-calmodulin) and methyl-4-azidobenzimidate. Azidocalmodulin containing one azido group per calmodulin retained its ability to stimulate the CA2+-sensitive phosphodiesterase purified from bovine heart muscle. The concentrations of calmodulin and azidocalmodulin required for half-maximal stimulation of phosphodiesterase activity were 170 and 230 pM, respectively. Azido-125I-calmodulin was used to photoaffinity label troponin I, myosin light chain kinase, and the Ca2+-sensitive phosphodiesterase. Formation of crosslinked complexes required the presence of Ca2+ or Mn2+ and was inhibited by excess unmodified calmodulin. The calmodulin-binding subunits all formed 1:1 complexes with calmodulin, and the molecular weights of the crosslinked products obtained with troponin I, the phosphodiesterase, and myosin light chain kinase were 43,000, 79,000, and 116,000, respectively. Photolysis experiments using azido-125I-calmodulin and bovine cerebral cortex membranes or detergent-solubilized membranes resulted in formation of a limited number of specifically labeled polypeptides. Azido-calmodulin appears to be an appropriate photoaffinity label for the identification and characterization of calmodulin-binding subunits.
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PMID:Preparation of azidocalmodulin: a photoaffinity label for calmodulin-binding proteins. 626 11


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