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PubMed1995

Modulation of the cGMP-gated ion channel in frog rods by calmodulin and an endogenous inhibitory factor.

1. Outer segment patches excised in the light were used to investigate the effects of exogenous calmodulin and an endogenous inhibitory factor on the cGMP-gated channel of frog rods. 2. Calmodulin shifted to the right the dose-response relation for activation of the channels by 8-Br-cGMP, but did not change the maximum current or the form of the relation. Reversal of this effect by removal of calmodulin was accelerated by brief exposure to saturating [8-Br-cGMP]. Inhibition by calmodulin required calcium and gave as much as a 5-fold decrease in current for an [8-Br-cGMP] functionally comparable to the presumed physiological [cGMP]. 3. Exposure to low [Ca2+]i (tens of nanomolar) appeared to irreversibly remove or inactivate an endogenous channel inhibitory factor from the patches, increasing the current at low [8-Br-cGMP]. Like calmodulin, this factor slowed the voltage-dependent channel-gating kinetics and did not change the maximum current. However, unlike calmodulin, the endogenous factor remained stably associated with the patches at high [Ca2+]i (1 microM), even with exposure to saturating [8-Br-cGMP]. 4. After the low-Ca2+ treatment increased the current, calmodulin reduced the current to about the same level as it had before the low-Ca2+ treatment, giving a larger fractional suppression. Furthermore, patches with high initial sensitivity to 8-Br-cGMP had small low-Ca2+ effects and large calmodulin effects, while the reverse was true for patches with low initial agonist sensitivity. 5. Application of trypsin to the intracellular surface of the patch prevented the responses to calmodulin and to low [Ca2+]i, suggesting involvement of a cytoplasmic portion of the channel. However, trypsin also reduced the total agonist-induced patch current. 6. Our results are consistent with a model in which calmodulin and an endogenous calcium-binding protein compete for the same site, inhibiting channel opening or cGMP binding. The tight association of the endogenous factor with the channel even at relatively low [Ca2+]i suggests that in the transducing rod it may inhibit the channels most of the time in darkness and in dim light, preventing any potential inhibitory effects of calmodulin. The endogenous factor would be expected to leave the channel only in bright or prolonged light, when the [Ca2+]i is thought to be very low.

Animals↗
PubMed1983

Pharmacological antagonism of calmodulin.

The widespread role of calmodulin in mediating Ca2+-dependent activities raises the possibility that calmodulin antagonists might be used effectively to alter cellular function. Current progress toward the development of calmodulin antagonists of suitable selectivity and specificity is considered in this review. Phenothiazines and other antipsychotic agents, including trifluoperazine, are among the most potent antagonists of calmodulin actions. Their antagonism of calmodulin function correlates with binding to calmodulin, probably at a hydrophobic region induced by Ca2+. Binding to this region is not stereospecific nor restricted to antipsychotic agents, as a wide range of hydrophobic molecules interact similarly and inhibit calmodulin activity. Trifluoperazine is not a specific antagonist of calmodulin, as it interacts with other Ca2+-binding proteins and also inhibits many lipid-dependent enzyme activities, including a widely distributed Ca2+ and phospholipid-dependent protein kinase. New data is presented showing that trifluoperazine, in addition to its binding to calmodulin, binds to the activated states of calmodulin-sensitive enzymes, in particular erythrocyte Ca2+- and Mg2+-stimulated ATPase. The implications of the lack of selectivity of presently available "calmodulin antagonists" are assessed.

Animals↗
PubMed1988

Phosphorylation of liver plasma membrane-bound calmodulin.

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [gamma-32P]ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3':5'AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Calmodulin mediates the stimulatory effect of 3,5,3'-triiodothyronine on adenylate cyclase activity in rat thymocyte plasma membranes.

We have previously demonstrated that T3 increases adenylate cyclase activity in rat thymocyte plasma membranes by a mechanism that is calcium dependent. In the present studies we have examined whether calmodulin participates in this response to T3. Initial experiments provided evidence that calmodulin is involved in regulating the activity of the guanyl nucleotide-dependent adenylate cyclase in this tissue. Thus, compared with findings in freshly prepared membranes, dialysis of thymocyte plasma membrane preparations for 20 h at 4 C decreased their calmodulin concentrations from an initial value of approximately 600 ng/100 micrograms protein by about 50% and decreased adenylate cyclase activity by approximately 80%. Although storage of the same preparations under comparable conditions resulted in no change in calmodulin content, it caused an approximate 30% decrease in adenylate cyclase activity. Addition of calmodulin had no effect on adenylate cyclase activity in fresh or stored membrane preparations, but produced a dose-dependent increase in enzyme activity in preparations that had been dialyzed. Further, when added to freshly isolated membranes, three calmodulin antagonists, trifluoperazine, calmidazolium, and calmodulin antibodies, all produced a concentration-dependent inhibition of adenylate cyclase activity, and this was completely reversed in all cases by the addition of high concentrations of exogenous calmodulin. The stimulation of guanyl nucleotide-dependent adenylate cyclase activity that T3 induced in fresh membrane preparations was present in membranes that had been stored, but was absent in those that had been dialyzed. In addition, the response to T3 in fresh membranes was inhibited or abolished by all three calmodulin antagonists. Both in dialyzed membranes and in the presence of antagonists, the response to T3 was restored by the addition of exogenous calmodulin. We conclude that calmodulin influences the activity of the guanyl nucleotide-dependent adenylate cyclase in rat thymocytes and ultimately mediates the stimulation of enzyme activity that T3 produces. Such mediation very likely explains the calcium-dependent nature of the stimulatory effect of T3 on thymocyte adenylate cyclase activity.

Adenylyl Cyclases↗
PubMed1997

Calmodulin localizes to the spindle pole body of Schizosaccharomyces pombe and performs an essential function in chromosome segregation.

The essential calmodulin genes in both Saccharomyces cerevisiae and Schizosaccharomyces pombe were precisely replaced with genes encoding fusions between calmodulin and the green fluorescent protein (GFP). In living budding yeast the GFP-calmodulin fusion protein (GFP-Cmd1p) localized simultaneously to sites of cell growth and to the spindle pole body (SPB), the yeast analog of the centrosome. Having demonstrated proper localization of GFP-calmodulin in budding yeast, we examined the localization of a fusion between GFP and calmodulin (GFP-Camlp) in fission yeast, where calmodulin had not been localized by any method. We find GFP-Camlp also localizes both to sites of polarized cell growth and to the fission yeast SPB. The localization of calmodulin to the SPB by GFP fusion was confirmed by indirect immunofluorescence. Antiserum to S. pombe calmodulin labeled the ends of the mitotic spindle stained with anti-tubulin antiserum. This pattern was identical to that seen using antiserum to Sad1p, a known SPB component. We then characterized the defects in a temperature-sensitive S. pombe calmodulin mutant. Mutant cam1-E14 cells synchronized in S phase completed DNA synthesis, but lost viability during transit of mitosis. Severe defects in chromosome segregation, including hypercondensation, fragmentation, and unequal allocation of chromosomal material were observed. Immunofluorescence analysis of tubulin revealed a population of cells containing either broken or mislocalized mitotic spindles, which were never observed in wild-type cells. Taken together with the subcellular localization of calmodulin, the observed spindle and chromosome segregation defects suggest that calmodulin performs an essential role during mitosis at the fission yeast SPB.

Calmodulin↗
PubMed1989

Topographical mapping of calmodulin-target enzyme interaction domains.

Calmodulin derivatives, specifically biotinylated in domains I and III, were synthesized to address the structures of calmodulin necessary for binding to its target enzymes in active conformations. By binding avidin to these biotinylated calmodulins, the role of specific sequences of the calmodulin molecule in target enzyme interactions could then be evaluated. The role of domain I in these interactions was assessed by biotinylation of Cys-27 of wheat germ calmodulin with N-ethylmaleimidobiotin. This modification did not affect the ability of this calmodulin to activate 3'-5'-cyclic nucleotide phosphodiesterase (PDE) or human erythrocyte Ca2+-Mg2+ ATPase. The addition of avidin to form a stable calmodulin-avidin complex also did not affect activation. Bovine testes calmodulin was biotinylated on Lys-94 by calcium-dependent reaction with N-hydroxysuccinimido ester-biotin at pH 6.0. This derivative was used to probe the Ca+2 binding region of domain III. The incorporation of biotin at Lys-94 of bovine calmodulin did not affect calmodulin activation of PDE. However, compared to unmodified calmodulin, a 4-fold higher concentration of this derivative was required to fully activate the ATPase. The addition of excess avidin to this derivative abolished all activation for both PDE and the ATPase. Sites of modification were determined by sequence analysis of labeled peptides.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1985

Differential trace labeling of calmodulin: investigation of binding sites and conformational states by individual lysine reactivities. Effects of beta-endorphin, trifluoperazine, and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid.

The Ca2+-dependent association of beta-endorphin and trifluoperazine with porcine testis calmodulin, as well as the effects of removing Ca2+ by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) treatment, were investigated by the procedure of differential kinetic labeling. This technique permitted determination of the relative rates of acylation of each of the epsilon-amino groups of the seven lysyl residues on calmodulin by [3H]acetic anhydride under the different conditions. In all cases, less than 0.52 mol of lysyl residue/mol of calmodulin was modified, thus ensuring that the labeling pattern reflects the microenvironments of these groups in the native protein. Lysines 75 and 94 were found to be the most reactive amino groups in Ca2+-saturated calmodulin. In the presence of Ca2+ and under conditions where beta-endorphin and calmodulin were present at a molar ratio of 2.5:1, the amino groups of lysines 75 and 148 were significantly reduced in reactivity compared to calmodulin alone. At equimolar concentrations of peptide and protein, essentially the same result was obtained except that the magnitudes of the perturbation of these two lysines were less pronounced. With trifluoperazine, at a molar ratio to calmodulin of 2.5:1, significant perturbations of lysines 75 and 148, as well as Lys 77, were also found. These results further substantiate previous observations of a commonality between phenothiazine and peptide binding sites on calmodulin. Lastly, an intriguing difference in Ca2+-mediated reactivities between lysines 75 and 77 of calmodulin is demonstrated. In the Ca2+-saturated form of the protein, both lysines are part of the long connecting helix between the two homologous halves of the protein (Babu, Y. S., Sack, J. S., Greenhough, T. G., Bugg, C. E., Means, A. R., and Cook, W. J. (1985) Nature 315, 37-40). Yet, Lys 75 increases in reactivity some 25-fold, compared to only a 2-fold change for Lys 77, in going from EGTA-treated to Ca2+-saturated calmodulin. Thus, the microenvironment of Lys 75 is markedly altered upon Ca2+ binding, and this linker region between the two globular lobes of the protein appears to be quite important in the interaction of calmodulin with inhibitory molecules and perhaps activatable enzymes.

Acetylation↗
PubMed1988

Inhibition of mitosis in PtK2 cells by CAPP1-calmodulin.

Various indirect evidence has indicated that calcium ions and the calcium-binding regulator protein, calmodulin, may regulate mitosis in higher eukaryotes. We have used the competitive antagonist, CAPP1-calmodulin, to antagonize intracellular calmodulin and test the hypothesis that calmodulin serves as a regulator of mitosis. We find that CAPP1-calmodulin inhibits the transit of cells through metaphase at estimated intracellular concentrations up to that of native calmodulin; beyond that level, the inhibition of mitosis vanishes. The membrane-permeant anticalmodulin agents, W7 and calmidazolium, also inhibit the progress of cells through metaphase. The similarity of the inhibitory curves for CAPP1-calmodulin, W7, and calmidazolium suggests that all these agents inhibit mitosis by antagonizing intracellular calmodulin. In order to test whether this inhibition of metaphase transit is due to an effect of the agents on intracellular free calcium, we used the calcium indicator Fura-2 to measure intracellular calcium levels after CAPP1-calmodulin injection or during calmidazolium treatment. We found that, while intracellular calcium levels are modestly elevated during calmidazolium treatment, they were unaffected by CAPP1-calmodulin, a result suggesting that mitosis inhibition was not due to an effect on intracellular free calcium. The reasons for the anomalous dose-response behavior of these drugs are not known; however, the behavior of cells at drug levels below the point of anomaly supports the hypothesis that calmodulin acts as a regulator of mitosis in these cells.

Analysis of Variance↗
PubMed1982

Calmodulin from neurospora crassa. General properties and conformational changes.

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.

Amino Acids↗
PubMed1984

Calmodulin binds to both microtubule-associated protein 2 and tau proteins.

Calmodulin binding to microtubule-associated proteins (MAPs) was studied by using three experimental techniques: affinity chromatography, cross-linking, and equilibrium binding. 1) Calmodulin affinity chromatography: both MAP2 and tau proteins were bound to calmodulin affinity columns in the presence of calcium and released with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), whereas tubulin was not bound. 2) Cross-linking 125I-calmodulin to whole MAPs and MAP2 by disuccinimidyl suberate: 125I-calmodulin was cross-linked to MAP2 and tau proteins showing an intense radioactivity band at 300,000 daltons and a diffuse band between 70,000 and 90,000 daltons. The cross-linking was calcium-dependent and was blocked by EGTA, trifluoperazine, or excess unlabeled calmodulin. 3) Equilibrium binding of 125I-calmodulin to MAP2 and tau using the Hummel-Dreyer technique on Sephadex G-100 columns: MAP2 and tau proteins bound 125I-calmodulin in a calcium-dependent manner and no binding occurred in the presence of EGTA. The apparent dissociation constant of calmodulin for MAP2 was 7 microM. The results indicate that calmodulin exerts its effect on microtubule assembly through the formation of a "Ca2+ X calmodulin X MAP2 or X tau" complex.

Animals↗
PubMed1980

Effects of lanthanum and trifluoperazine on [125I]calmodulin binding to rat striatal particulates.

A calmodulin-binding assay was established in rat striatal particulates which were depleted of endogenous calcium and calmodulin by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) treatment. The binding of 125I-labeled calmodulin to this preparation was saturable and time-dependent. The dependence of calmodulin binding upon temperature and concentration was also demonstrated. Calcium was a prerequisite for calmodulin binding and it facilitated the binding in a dose-dependent manner. Lanthanum, a known calcium antagonists in other tissue systems, mimicked the effect of calcium on calmodulin binding. When both ions were present at low concentrations, their effects on calmodulin binding was additive. Lanthanum, but not calcium, inhibited calmodulin release from a non-EGTA-treated preparation. This difference in action between calcium and lanthanum suggests that they mediate calmodulin binding in an independent manner. Scatchard analysis of calcium-calmodulin binding to rat striatal particulates revealed that there are two populations of binding sites: a higher affinity (apparent KD = 1.3 X 10(-7) M) and a lower affinity (apparent KD = 2.9 X 10(-7) M) binding site. Trifluoperazine, a phenothiazine antipsychotic drug, at 10(-4) M antagonized calmodulin binding only at the higher affinity binding sites. These sites may play an important role in mediating the action of trifluoperazine in the caudate nucleus.

Animals↗
PubMed1993

Ca2+ calmodulin regulates basal gallbladder absorption.

BACKGROUND: Gallbladder absorption is altered during gallstone formation, a phenomenon that may be partly the result of elevated biliary Ca2+ levels. Recent studies suggest that changes in gallbladder absorption are mediated by intracellular Ca2+ ([Ca2+]ic). However, the mechanisms by which [Ca2+]ic regulates gallbladder ion transport are not known. Calmodulin is a Ca2+ receptor protein in the Ca2+ messenger system that modulates ion transport in the small intestine. We hypothesized that Ca(2+)-calmodulin mediates the effects of [Ca2+]ic on gallbladder absorption. METHODS: Prairie dog gallbladders were mounted in Ussing chambers, and standard electrophysiologic parameters were recorded. Unidirectional Na+, Cl-, and water fluxes were measured before and after mucosal exposure to 5 x 10(-5) mol/L trifluoperazine, a potent calmodulin antagonist. In addition, the ion transport effects of increased extracellular calcium and theophylline were determined in the presence of calmodulin inhibition. RESULTS: Inhibition of calmodulin resulted in an increase in net Na+ and water absorption and converted the gallbladder from a Cl- absorptive state. Similar results were obtained during exposure to two other calmodulin antagonists that differ only in their affinity for calmodulin but not in their hydrophobicity, suggesting that the observed changes were caused by specific calmodulin inhibition. Effects of trifluoperazine were reversed by increasing luminal [Ca2+] or theophylline exposure. CONCLUSIONS: The effects of calmodulin inhibition are directly opposite of the effects of the Ca2+ ionophore. We conclude that Ca(2+)-calmodulin regulates gallbladder absorption at basal [Ca2+]ic. Further studies are needed to determine whether altered calmodulin activity is responsible for increased gallbladder absorption during gallstone formation.

Absorption↗
PubMed2004

Volume-regulated anion conductance in cultured rat cerebral astrocytes requires calmodulin activity.

We examined the calmodulin dependence of anion channel activation during hypo-osmotic swelling in rat cerebral astrocytes. Control cells bathed in iso-osmotic (290 mOsm) phosphate-buffered saline (PBS) and recorded using a patch electrode containing 140 mM KCl increased membrane conductance threefold over basal levels after 12 min in hypo-osmotic (200 mOsm) PBS. Cells injected with monoclonal anticalmodulin antibody demonstrated no increase in membrane conductance during a subsequent exposure to hypo-osmotic PBS. In contrast, cells iontophoretically injected with monoclonal antiglial fibrillary acidic protein antibody or with anticalmodulin antibody absorbed with an excess of free calmodulin demonstrated an increase in conductance during hypo-osmotic exposure similar to that of control cells. Conductance in iso-osmotic conditions was unchanged by antibody injection. Similar results were obtained when using patch electrode and bath solutions containing chloride as the only cell permeant ion, indicating a calmodulin-dependent anion current is activated with this degree of hypo-osmotic treatment. Western blots confirmed the specificity of the anticalmodulin and antiglial fibrillary acidic protein antibodies used in this study for proteins of 17 and 51 kD, respectively. In addition, in vitro studies demonstrated inhibition of the calmodulin-dependent activation of phosphodiesterase by the anticalmodulin antibody. Thus, binding of this antibody to calmodulin causes functional inhibition of calmodulin activity. No change in the intensity or cellular distribution of calmodulin immunostaining was observed during 30 min of hypo-osmotic exposure. However, increased immunostaining for activated calmodulin kinase IIalpha was observed after 10 min of hypo-osmotic exposure, suggesting initiation of calmodulin-dependent processes by cell swelling. The data indicate calmodulin activity is critical for activation of volume-regulated anion channels in rat cerebral astrocytes.

Animals↗
PubMed1997

Functional consequences of truncating amino acid side chains located at a calmodulin-peptide interface.

To test the relevance of the calmodulin-peptide crystal structures to their respective calmodulin-enzyme interactions, amino acid side chains in calmodulin were altered at positions that interact with the calmodulin-binding peptide of smooth muscle myosin light chain kinase but not with the calmodulin kinase IIalpha peptide. Since shortening the side chains of Trp-800, Arg-812, and Leu-813 in smooth muscle myosin light chain kinase abrogated calmodulin-dependent activation (Bagchi, I. C., Huang, Q., and Means, A. R. (1992) J. Biol. Chem. 267, 3024-3029), substitutions were introduced at positions in calmodulin which contact residues corresponding to Arg-812 and Leu-813 in the smooth muscle myosin light chain kinase peptide. Assays of smooth muscle myosin light chain kinase with the calmodulin mutants M51A,V55A, L32A,M51A,V55A, and L32A,M51A,V55A,F68L, M71A exhibited 60%, 25%, and less than 1% of maximal activity respectively, whereas the mutants fully activated calmodulin kinase IIalpha. Alanine substitutions at positions on the smooth muscle myosin light chain kinase peptide, corresponding to Trp-800 and Arg-812 in the enzyme, produced an 8-fold increase in the enzyme inhibition constant in contrast with the abolition of calmodulin binding by similar mutations in the parent enzyme.

Amino Acids↗
PubMed1998

Substrate-directed function of calmodulin in autophosphorylation of Ca2+/calmodulin-dependent protein kinase II.

Autophosphorylation of Thr286 in Ca2+/calmodulin-dependent protein kinase II occurs within each holoenzyme by an intersubunit reaction and is essential for kinase function in vivo. In addition to a kinase-directed function of calmodulin to activate the kinase, a second calmodulin is required for the autophosphorylation of each Thr286 (Hanson, P. I., Meyer, T., Stryer, L., and Schulman, H. (1994) Neuron 12, 943-956). We have engineered heteromeric holoenzymes comprising distinct "kinase" and "substrate" subunits to test for kinase- and substrate-directed functions of calmodulin. The obligate kinase subunits have aspartate residues substituted for threonine at positions 286, 305, and 306 (the autophosphorylation and calmodulin-binding sites), making it constitutively active but unable to bind calmodulin. Obligate substrate subunits are catalytically inactive (K42M mutation) but are able to bind calmodulin. Phosphorylation of substrate subunits occurs specifically at Thr286 and is completely dependent upon the presence of calmodulin. Blocking the ability of the substrate subunit to bind calmodulin, either with inhibitor KN-93 or by mutagenesis of the calmodulin-binding domain of the substrate subunit, prevents its phosphorylation, consistent with a substrate-directed function of calmodulin that requires its direct binding to the subunit being phosphorylated.

Calcium Signaling↗
PubMed1984

Structural characterization of a higher plant calmodulin : spinacia oleracea.

Calmodulin is a eukaryotic calcium binding protein which has several calcium-dependent in vitro activities. Presented in this report is a structural characterization of calmodulin from spinach leaves (Spinacia oleracea). Spinach calmodulin may be representative of higher plant calmodulins in general since calmodulin from the monocotyledon barley (Hordeum vulgare) is indistinguishable by a variety of physical, chemical, and functional criteria (Schleicher, Lukas, Watterson 1983 Plant Physiol 73: 666-670). Spinach calmodulin is homologous to bovine brain calmodulin with only 13 identified amino acid sequence differences, excluding a blocked NH(2)-terminal tripeptide whose sequence has not been elucidated. Two extended regions of sequence identity are in the NH(2)-terminal half of the molecule, while nine of the 13 identified differences are in the COOH-terminal half of the molecule. Two of the changes, a cysteine at residue 26 and a glutamine at residue 96, require a minimum of two base changes in the nucleotide codons. Both of these changes occur in the proposed calcium binding loops of the molecule. Five additional amino acid differences found in spinach calmodulin had not been observed previously in a calmodulin. As described in an accompanying report (Roberts, Burgess, Watterson 1984 Plant Physiol 75: 796-798), these limited number of amino acid sequence variations appear to result in differential effects on the activation of calmodulin-dependent enzymes by plant and vertebrate calmodulins.

Journal Article↗
PubMed1984

Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins.

In the preceding paper (Lukas, Iverson, Schleicher, Watterson 1984 Plant Physiol 75: 788-795), we reported that the amino acid sequence of spinach calmodulin has at least 13 amino acid sequence differences from vertebrate calmodulin. In the present study, we investigated the effect of these amino acid sequence substitutions on the enzyme activator properties of vertebrate and plant calmodulins. Calmodulins from spinach and the green alga Chlamydomonas reinhardtii activate chicken gizzard myosin light chain kinase in a manner similar but not identical to chicken calmodulin. In contrast, these calmodulins have very different NAD kinase activator properties. The concentration required for half-maximal activation of pea seedling NAD kinase by spinach calmodulin (3-4 nanomolar) is lower than the corresponding concentrations of chicken (20 nanomolar) and Chlamydomonas (40 nanomolar) calmodulins. However, the maximum level of activation obtained with Chlamydomonas calmodulin is 4- to 6-fold higher than spinach or chicken calmodulin. These data indicate that the limited structural heterogeneity among calmodulins have differential effects on their biochemical activities.

Journal Article↗
PubMed1985

Role of calmodulin inhibition in the mode of action of ophiobolin a.

Calmodulin has been isolated from the root of Zea mays. It activates the bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase and has electrophoretic mobility very similar to that of bovine brain calmodulin. Ophiobolin A, a fungal toxin, interacts with the maize calmodulin. The interaction is not reversed by dilution or denaturation in SDS and results in the loss of ability of the calmodulin to activate the phosphodiesterase. The inhibition is much faster in the presence than in the absence of Ca(2+). The electrophoretic mobility of ophiobolin A-treated calmodulin is less than that of untreated calmodulin. Several similarities are found between the inhibition of maize calmodulin by ophiobolin A in vitro and the effects of ophiobolin A on excised roots. Both are irreversible and time-dependent. The concentration of ophiobolin A for half-maximal inhibition of calmodulin in the phosphodiesterase assay is similar to that for phytotoxicity. In both cases ophiobolin A derivatives behave similarly, i.e. 18-bromo-19-methoxyophiobolin A is as potent as ophiobolin A, while 3-anhydro-ophiobolin A and 6-epi-ophiobolin A are less potent. A smaller amount of active calmodulin was measured in the extract from ophiobolin A-treated roots than in those from untreated roots. The present study suggests that calmodulin is a target molecule in the root for the toxicity of ophiobolin A.

Journal Article↗