Addition Of Exogenous Calmodulin Research Articles

Ca2+‐calmodulin interacts with DdCAD‐1 and promotes DdCAD‐1 transport by contractile vacuoles in Dictyostelium cells

The Ca(2+) -dependent cell-cell adhesion molecule DdCAD-1, encoded by the cadA gene of Dictyostelium discoideum, is synthesized at the onset of development as a soluble protein and then transported to the plasma membrane by contractile vacuoles. Calmodulin associates with contractile vacuoles in a Ca(2+) -dependent manner, and co-localizes with DdCAD-1 on the surface of contractile vacuoles. Bioinformatics analysis revealed multiple calmodulin-binding motifs in DdCAD-1. Co-immunoprecipitation and pull-down studies showed that only Ca(2+) -bound calmodulin was able to bind DdCAD-1. Structural integrity of DdCAD-1, but not the native conformation, was required for its interaction with calmodulin. To investigate the role of calmodulin in the import of DdCAD-1 into contractile vacuoles, an in vitro import assay consisting of contractile vacuoles derived from cadA(-) cells and recombinant proteins was employed. Prior stripping of the bound calmodulin from contractile vacuoles by EGTA impaired import of DdCAD-1, which was restored by addition of exogenous calmodulin. The calmodulin antagonists W-7 and compound 48/80 blocked the binding of calmodulin onto stripped contractile vacuoles, and inhibited the import of DdCAD-1. Together, the data show that calmodulin forms a complex with DdCAD-1 and promotes the docking and import of DdCAD-1 into contractile vacuoles. CaM physically interacts with DdCAD-1 by pull down (View Interaction: 1, 2) DdCAD-1 binds to CaM by far western blotting (View interaction) DdCAD-1 physically interacts with CaM by anti bait coimmunoprecipitation (View interaction).

Defective Interaction of Calmodulin With RyR2 Plays a Key Role in the Pathogenesis of Leaky Channel in Failing Hearts

Background: We previously reported that interaction between N-terminal1-600 and central domains2000-2500 of ryanodine receptor (RyR2) is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca2+ release in pacing-induced failing hearts. Methods & Results: Cardiomyocytes were isolated from dog LV muscles {normal; 4-weeks rapid RV pacing (heart failure) }. To assess CaM binding to RyR2, sarcoplasmic reticulum (SR) was mixed with CaM-SANPAH conjugate (16 nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR. In normal SR, the decreased CaM binding seen in failing SR was reproduced by addition of FK506, which was found to induce domain unzipping mimicking failing SR. In normal cardiomyocytes, Ca2+ spark frequency (CaSF) was markedly increased by adding FK506, but this increase was inhibited by addition of exogenous CaM. In failing cardiomyocytes, CaSF was higher than normal at baseline, but it was again reduced by exogenous CaM. Conclusion: The defective inter-domain interaction within RyR2 seems to increase spontaneous Ca2+ release events in failing SR, via the reduced CaM-binding to RyR2.

Calcium Regulation of Calmodulin Binding to and Dissociation from the Myo1c Regulatory Domain

Myo1c is an unconventional myosin involved in cell signaling and membrane dynamics. Calcium binding to the regulatory-domain-associated calmodulin affects myo1c motor properties, but the kinetic details of this regulation are not fully understood. We performed actin gliding assays, ATPase measurements, fluorescence spectroscopy, and stopped-flow kinetics to determine the biochemical parameters that define the calmodulin-regulatory-domain interaction. We found calcium moderately increases the actin-activated ATPase activity and completely inhibits actin gliding. Addition of exogenous calmodulin in the presence of calcium fully restores the actin gliding rate. A fluorescently labeled calmodulin mutant (N111C) binds to recombinant peptides containing the myo1c IQ motifs at a diffusion-limited rate in the presence and absence of calcium. Measurements of calmodulin dissociation from the IQ motifs in the absence of calcium show that the calmodulin bound to the IQ motif adjacent to the motor domain (IQ1) has the slowest dissociation rate (0.0007 s-1), and the IQ motif adjacent to the tail domain (IQ3) has the fastest dissociation rate (0.5 s-1). When the complex is equilibrated with calcium, calmodulin dissociates most rapidly from IQ1 (60 s-1). However, this increased rate of dissociation is limited by a slow calcium-induced conformational change (3 s-1). Fluorescence anisotropy decay of fluorescently labeled N111C bound to myo1c did not depend appreciably on Ca2+. Our data suggest that the calmodulin bound to the IQ motif adjacent to the motor domain is rapidly exchangeable in the presence of calcium and is responsible for regulation of myo1c ATPase and motile activity.

Ca 2+/calmodulin binds and dissociates K-RasB from membrane

We have investigated the interaction of calmodulin (CaM) with Ras-p21 and the significance of this association. All Ras-p21 isoforms tested (H-, K-, and N-Ras) were detected in the particulate fraction of human platelets and MCF-7 cells, a human breast cancer cell line. In MCF-7 cells, H- and N-Ras were also detected in the cytosolic fraction. K-RasB from platelet and MCF-7 cell lysates was found to bind CaM in a Ca 2+-dependent but GTPγS-independent manner. The yeast two-hybrid analysis demonstrated that K-RasB binds to CaM in vivo. Incubation of isolated membranes from platelet and MCF-7 cells with CaM caused dissociation of only K-RasB from membranes in a Ca 2+-dependent manner. CaM antagonist, W7, inhibited dissociation of K-RasB. Addition of platelet or MCF-7 cytosol alone to isolated platelet membranes did not cause dissociation of K-RasB and only addition of exogenous CaM caused dissociation. The results suggest a potential role for Ca 2+/CaM in the regulation of K-RasB function.

Isolation and characterization of 190K protein from aorta smooth muscle.

Molecular assemblies of actin and myosin for the contractility of smooth muscle are quite different from those of striated muscle. Another striking difference is that vascular smooth muscle has a potential to transform to migratory synthetic cell type. At this point of view, smooth muscle cell has properties similar to those of non-muscle. In fact, myosin Ic, a single headed unconventional myosin, was identified in aorta smooth muscle. During the studies on myosin Ic, we have found another calmodulin related 190kDa protein. This protein binds to calmodulin irrespective on calcium ion and to F-actin in an ATP independent manner. Furthermore, the F-actin binding stoichiometry diminished to half upon the addition of exogenous calmodulin. Partial amino acid sequence indicated a high homology to those of GRD (GTPase Related Domain) of human brain IQGAP1. Western blot analysis using anti-human IQGAP1 antibody also indicated a strong cross-reactivity with the protein. We have tested the protein with respect to the characteristic F-actin gelation by IQGAP1. In the presence of cdc42 and GTPgammaS, 190kDa protein could cause a high viscosity of F-actin. These data indicate a close similarity to human brain IQGAP1. The presence of IQGAP1 in aorta smooth muscle suggests contributions for cellular processes such as actin reorganization during contraction-relaxation cycle, association of cytoskeletal structure to cell membrane, organelle movement.

Ca2+-dependent Regulation of the Motor Activity of Myosin V

Mouse myosin V constructs were produced that consisted of the myosin motor domain plus either one IQ motif (M5IQ1), two IQ motifs (M5IQ2), a complete set of six IQ motifs (SHM5), or the complete IQ motifs plus the coiled-coil domain (thus permitting formation of a double-headed structure, DHM5) and expressed in Sf9 cells. The actin-activated ATPase activity of all constructs except M5IQ1 was inhibited above pCa 5, but this inhibition was completely reversed by addition of exogenous calmodulin. At the same Ca(2+) concentration, 2 mol of calmodulin from SHM5 and DHM5 or 1 mol of calmodulin from M5IQ2 were dissociated, suggesting that the inhibition of the ATPase activity is due to dissociation of calmodulin from the heavy chain. However, the motility activity of DHM5 and M5IQ2 was completely inhibited at pCa 6, where no dissociation of calmodulin was detected. Inhibition of the motility activity was not reversed by the addition of exogenous calmodulin. These results indicate that inhibition of the motility is due to conformational changes of calmodulin upon the Ca(2+) binding to the high affinity site but is not due to dissociation of calmodulin from the heavy chain.

Pharmacological and immunohistochemical characterization of calmodulin-stimulated (Ca(2+)+Mg(2+))-ATPase in cultured porcine aortic endothelial cells.

Plasma membrane (Ca(2+)+Mg(2+))-ATPase and Ca(2+) transport activities, best characterized in human erythrocytes, are stimulated by calmodulin and thought to play a crucial role in the termination of cellular Ca(2+) signaling in all cells. In plasma membranes isolated from cultured porcine aortic endothelial cells, the (Ca(2+)+Mg(2+))-ATPase was not readily measured. This is in part because of an overabundance of nonspecific Ca(2+)- and/or Mg(2+)-activated ecto-5'-nucleotide phosphohydrolases. Moreover, addition of exogenous calmodulin (10(-9) to 10(-6) mol/L) produced no measurable stimulation of ATPase activities, suggesting a permanently activated state or, alternatively, a complete lack thereof. To establish and verify the presence of a calmodulin-regulated (Ca(2+)+Mg(2+))-ATPase activity in these endothelial cells, immunohistochemical localization using a monoclonal mouse anti-(Ca(2+)+Mg(2+))-ATPase antibody (clone 5F10) was applied to intact pig aorta endothelium, cultured endothelial monolayers, and isolated endothelial plasma membrane fractions. This approach clearly demonstrated Ca(2+) pump immunoreactivity in each of these preparations. To confirm functional calmodulin stimulation of the (Ca(2+)+Mg(2+))-ATPase, 10(-5) mol/L calmidazolium (R24571) was added to the isolated plasma membrane preparation, which lowered the (Ca(2+)+Mg(2+))-ATPase activity from 143.0 to 78.15 nmol P(i)/mg protein x min(-1). This calmidazolium-reduced activity could then be stimulated 113.1+/-0.8% in a concentration-dependent manner by the addition of exogenous calmodulin (10(-7) to 2 x 10(-6) mol/L) with an EC(50) of 3.45+/-0.04 x 10(-7) mol/L (n=4). This represents a competitive lowering of the apparent calmodulin affinity by approximately 100 compared with other unopposed calmodulin-stimulated processes. Together, these findings support evidence for the presence of a calmodulin-stimulated plasma membrane (Ca(2+)+Mg(2+))-ATPase activity in cultured porcine aortic endothelial cells.

Biochemical evidence for a calmodulin-stimulated calcium-dependent protein kinase in maize.

We provide biochemical evidence for the presence of a Ca2+-dependent calmodulin (CaM)-stimulated protein kinase (CCaMK) from etiolated maize coleoptiles. The kinase, with a molecular mass of 72.3 kDa, was purified to homogeneity by means of ammonium sulphate precipitation, DEAE-Sephacel chromatography. CaM-Sepharose chromatography and gel purification. The purified kinase required 5 mM Mg2+ for activity and had an optimum pH of 7.5. The kinase is a Ca2+-binding protein, as was evident by 45Ca2+-binding and Ca2+ mobility-gel-shift assays. 1 microM Ca2+ stimulated the kinase activity about 12-fold and was further stimulated by the addition of exogenous CaM (approximately 100 nM). Addition of Ca2+ and CaM antagonists decreased the kinase activity. Under in vitro assay conditions the kinase phosphorylated preferentially syntide-2, histone IIIS and casein. Syntide-2 and histone IIIS were phosphorylated at serine residues, showing that the kinase belongs to the serine/threonine family of protein kinases. Autophosphorylation of CCaMK occurred on threonine residue(s) and was Ca2+ dependent. Addition of exogenous CaM had no effect on autophosphorylation. The properties of the maize kinase suggests that it is a CCaMK that shows dual stimulation with Ca2+ and CaM for substrate phosphorylation and only Ca2+ requirement for autophosphorylation. Antibodies raised against the kinase cross-reacted with maize total proteins to give a single band of 72 kDa and precipitated substrate (syntide-2 and histone IIIS)-phosphorylation and autophosphorylation activities in a specific manner. Localisation studies with antibodies showed that the kinase is ubiquitous.

Caveolin versus calmodulin. Counterbalancing allosteric modulators of endothelial nitric oxide synthase.

Nitric oxide is synthesized in diverse mammalian tissues by a family of calmodulin-dependent nitric oxide synthases. The endothelial isoform of nitric oxide synthase (eNOS) is targeted to the specialized signal-transducing membrane domains termed plasmalemmal caveolae. Caveolin, the principal structural protein in caveolae, interacts with eNOS and leads to enzyme inhibition in a reversible process modulated by Ca2+-calmodulin (Michel, J. B., Feron, O., Sacks, D., and Michel, T. (1997) J. Biol. Chem. 272, 15583-15586). Caveolin also interacts with other structurally distinct signaling proteins via a specific region identified within the caveolin sequence (amino acids 82-101) that appears to subserve the role of a "scaffolding domain." We now report that the co-immunoprecipitation of eNOS with caveolin is completely and specifically blocked by an oligopeptide corresponding to the caveolin scaffolding domain. Peptides corresponding to this domain markedly inhibit nitric oxide synthase activity in endothelial membranes and interact directly with the enzyme to inhibit activity of purified recombinant eNOS expressed in Escherichia coli. The inhibition of purified eNOS by the caveolin scaffolding domain peptide is competitive and completely reversed by Ca2+-calmodulin. These studies establish that caveolin, via its scaffolding domain, directly forms an inhibitory complex with eNOS and suggest that caveolin inhibits eNOS by abrogating the enzyme's activation by calmodulin.

Characterization of Human Liver Inducible Nitric Oxide Synthase Expressed inEscherichia coli

We have cloned the human liver inducible isoform of nitric oxide synthase (NOS) into anEscherichia coliexpression vector and have expressed and purified the enzyme. The protein has been expressed with and without a polyhistidine tail. In both cases, expression of functional protein requires coexpression with calmodulin and inclusion of tetrahydrobiopterin (H4B) in the purification buffers. Unlike the constitutive isoforms of NOS, this isoform is unstable in the absence ofl-arginine (l-Arg) and H4B toward loss of the heme group and the formation of a low-spin species spectroscopically distinct from that of the cofactor-bound protein. The enzyme purified in the presence of bothl-Arg and H4B is highly active, with aVmaxof ∼800 nmol NO min−1mg−1and aKmforl-Arg of 22 μm. The cytochrome c reductase activity is 38,000 nmol·min−1mg−1. Similar values are obtained for the enzyme with and without the polyhistidine tail. Ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid does not inhibit the activity of the protein, nor is the activity of the enzyme increased by the addition of exogenous calmodulin and/or Ca2+. These findings contrast with an earlier report, based on experiments with extracts of COS-1 cells expressing the recombinant enzyme, that the enzyme responds to changes in the Ca2+concentration. The human hepatic isoform is similar in its properties to the inducible NOS isoform purified from macrophages.

ATPase activity of myosin in hair bundles of the bullfrog's sacculus

Mechanoelectrical transduction by a hair cell displays adaptation, which is thought to occur as myosin-based molecular motors within the mechanically sensitive hair bundle adjust the tension transmitted to transduction channels. To assess the enzymatic capabilities of the myosin isozymes in hair bundles, we examined the actin-dependent ATPase activity of bundles isolated from the bullfrog's sacculus. Separation of 32P-labeled inorganic phosphate from unreacted [gamma-32P]ATP by thin-layer chromatography enabled us to measure the liberation of as little as 0.1 fmol phosphate. To distinguish the Mg(2+)-ATPase activity of myosin isozymes from that of other hair-bundle enzymes, we inhibited the interaction of hair-bundle myosin with actin and determined the reduction in ATPase activity. N-ethylmaleimide (NEM) decreased neither physiologically measured adaptation nor the nucleotide-hydrolytic activity of a 120-kDa protein thought to be myosin 1 beta. The NEM-insensitive, actin-activated ATPase activity of myosin increased from 1.0 fmol x s-1 in 1 mM EGTA to 2.3 fmol x s-1 in 10 microM Ca2+. This activity was largely inhibited by calmidazolium, but was unaffected by the addition of exogenous calmodulin. These results, which indicate that hair bundles contain enzymatically active, Ca(2+)-sensitive myosin molecules, are consistent with the role of Ca2+ in adaptation and with the hypothesis that myosin forms the hair cell's adaptation motor.

Isolation and identification of myosin I from porcine aorta media smooth muscle.

A complex of 110-kDa heavy chain and calmodulin was isolated from porcine aorta media smooth muscle and identified as myosin I. The isolated myosin I consisted of equimolar amounts of 110-kDa heavy chain and calmodulin. The addition of exogenous calmodulin to the complex revealed that a maximum of two molecules of calmodulin could be bound to the heavy chain. Isolated complex bound to F-actin in an ATP-dependent manner and its Mg(2+)-ATPase activity was activated by F-actin. In addition, it bound to phospholipid, which is a characteristic property of myosin I. Calcium ions induced a structural change, which was revealed by a difference in the cleavage pattern and for rate of cleavage by alpha-chymotrypsin. This behavior was similar to that reported for brush border myosin I [L.M. Collins and A. Bretscher (1988) J. Cell. Biol. 106, 367-373]. Calcium-dependent structural change of a complex of 110-kDa heavy chain and calmodulin was found from its solubility change at various NaCl concentrations in the presence of ATP. A complex of 116-kDa heavy chain and calmodulin, possibly another type of myosin I, was also isolated. A polyclonal antibody against the complex of 110-kDa heavy chain and calmodulin did not recognize the 116-kDa heavy chain. This result suggests that at least two types of myosin Is may exist at the protein level in porcine aorta media smooth muscle.

Activation of a Recombinant Petunia Glutamate Decarboxylase by Calcium/Calmodulin or by a Monoclonal Antibody Which Recognizes the Calmodulin Binding Domain

To date, only plants have been shown to possess a form of glutamate decarboxylase (GAD) that binds calmodulin. In the present study, a recombinant calmodulin-binding 58-kDa petunia GAD produced in Escherichia coli was purified to homogeneity using calmodulin-affinity chromatography, and its responsiveness to calcium and calmodulin was examined in vitro. At pH 7.0-7.5, the purified recombinant enzyme was essentially inactive in the absence of calcium and calmodulin, but it could be stimulated to high levels of activity (Vmax = 30 micromol of CO2 min-1 mg of protein-1) by the addition of exogenous calmodulin (K0.5 = 15 nM) in the presence of calcium (K0.5 = 0.8 microM). Neither calcium nor calmodulin alone had any effect on GAD activity. Recombinant GAD displayed hyperbolic kinetics at pH 7.3 (Km = 8.2 mM). A monoclonal antibody directed against the carboxyl-terminal region, which contains the calmodulin-binding domain of GAD, was able to fully activate GAD in a dose-dependent manner in the absence of calcium and calmodulin, whereas an antibody recognizing an epitope outside of this region was unable to activate GAD. This study provides the first evidence that the activity of the purified 58-kDa GAD polypeptide is essentially calcium/calmodulin-dependent at physiological pH. Furthermore, activation of GAD by two different proteins that interact with the calmodulin-binding domain, a monoclonal antibody or calcium/calmodulin, suggests that this domain plays a major role in the regulation of plant GAD activity.

Involvement of altered cytoskeletal protein phosphorylation in aluminum-induced CNS dysfunction.

In the present investigation, we have studied the effects of aluminum (10 mg/kg of body weight per day, i.p.) and desferrioxamine (6 mg/kg of body weight per day, i.p.) alone and in combination for 4 weeks on the regulation of phosphorylation of neuronal proteins. A marked decrease in the biological activity of calmodulin was observed after aluminum treatment; however, in combination with desferrioxamine, a reversal in the levels of calmodulin, in terms of nmol cAMP hydrolyzed/min/mg protein, was observed. Exogenous addition of calmodulin had an inhibitory effect on calmodulin-mediated synaptosomal protein phosphorylation in aluminum-exposed animals. An almost complete reversal of this inhibition was observed following coexposure to aluminum and desferrioxamine. Cyclic AMP-dependent synaptosomal protein phosphorylation was, however, stimulated following aluminum exposure. Coadministration of desferrioxamine along with aluminum was found to mitigate the neurotoxic effect of aluminum.

Alterations in calcium homeostasis on lead exposure in rat synaptosomes.

The effects of lead on Ca2+ homeostasis in nerve terminals was studied. Incubation with lead in vitro stimulated the activity of calmodulin and the maximum effect was observed at 30 microM lead, higher concentrations had an inhibitory effect. In vivo exposure to lead increased the activity of calmodulin by 45%. Lead had an inhibitory effect on Ca2+ ATPase activity in both calmodulin-rich and calmodulin-depleted synaptic plasma membranes, the IC50 values for inhibition being 13.34 and 16.69 microM respectively. Exogenous addition of calmodulin (5 micrograms) and glutathione (1 mM) to calmodulin rich synaptic plasma membranes reversed the inhibition by IC50 concentration of lead. In vivo exposure of lead also significantly reduced the Ca2+ ATPase activity, resulting in an increase in intrasynaptosomal calcium. Concomitant with the increase in intrasynaptosomal calcium, lipid peroxidation values also increased significantly in lead-treated animals. In addition lead also had an inhibitory effect on depolarization induced Ca2+ uptake and the inhibition was found to be a competitive one. The results suggest that lead exerts its toxic effects by modifications of the intracellular calcium messenger system which would have serious consequences on neuronal functioning.

Calmodulin-dependency of a Ca 2+ -pump at the plasma membrane of cultured carrot cells

Activity of a Ca 2+ -translocating ATPase which functions as a Ca 2+ -pump was found to be associated with a highly purified plasma membrane fraction of cultured carrot cells. Different classes of calmodulin antagonists — trifluoperazine and W-7 — appreciably inhibited ATP-driven uptake of Ca 2+ into the sealed vesicles of the membrane. The incorporation of Ca 2+ was also markedly inhibited in the presence of anti-calmodulin IgG. This translocating activity was significantly decreased when the vesicles were washed with EGTA-containing buffer; however, it was restored to almost the control level upon the addition of exogenous calmodulin. These results suggest that the Ca 2+ -pumping ATPase at the plasma membrane of cultured carrot cells is regulated by calmodulin, and the modulator protein associates with the enzyme in a Ca 2+ -dependent manner.

Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry

We examined the Ca(2+)-dependent regulation of brush border (BB) myosin-I by probing the possible roles of the calmodulin (CM) light chains. BB myosin-I MgATPase activity, sensitivity to chymotryptic digestion, and mechanochemical properties were assessed using 1-10 microM Ca2+ and in the presence of exogenously added CM since it has been proposed that this myosin is regulated by calcium-induced CM dissociation from the 119-kD heavy chain. Each of these BB myosin-I properties were dramatically altered by the same threshold of 2-3 microM Ca2+. Enzymatically active NH2-terminal proteolytic fragments of BB myosin-I which lack the CM binding domains (the 78-kD peptide) differ from CM-containing peptides in that the former is completely insensitive to Ca2+. Furthermore, the 78-kD peptide exhibits high levels of MgATPase activity which are comparable to that observed for BB myosin-I in the presence of Ca2+. This suggests that Ca2+ regulates BB myosin-I MgATPase by binding directly to the CM light chains, and that CM acts to repress endogenous MgATPase activity. Ca(2+)-induced CM dissociation from BB myosin-I can be prevented by the addition of exogenous CM. Under these conditions Ca2+ causes a reversible slowing of motility. In contrast, in the absence of exogenous CM, motility is stopped by Ca2+. We demonstrate this reversible slowing is not due to the presence of inactive BB myosin-I molecules exerting a "braking" effect on motile filaments. However, we did observe Ca(2+)-independent slowing of motility by acidic phospholipids, suggesting that factors other than Ca2+ and CM content can affect the mechanochemical properties of BB myosin-I.

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

Inhibition of protein synthesis by antagonists of calmodulin in Ehrlich ascites tumor cells

Several recent publications indicate that Ca2+ is required for protein synthesis in mammalian cells, including the Ehrlich ascites tumor cell. The present communication examines whether the effects of Ca2+ might be mediated through calmodulin or a related protein. Four calmodulin antagonists belonging to different chemical categories were used to provide evidence of calmodulin involvement. Three of the antagonists inhibited protein synthesis in intact cells; 50% inhibitory concentrations were 10 microM calmidazolium, 12 microM N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W7) and 17.5 microM trifluoperazine (TFP). Initiation was preferentially inhibited as indicated by an increase in the 80S monomers accompanied by a significant disaggregation of polyribosomes. All the antagonists also inhibited protein synthesis initiation in the cell-free protein-synthesizing system; 50% inhibitory concentrations for compound 48/80, calmidazolium, TFP, and W7 were 10 microM, 125 microM, 300 microM and 500 microM, respectively. A weak analogue of W7 inhibited only 20% at 1000 microM. Inhibition in the cell-free system was reversed by the addition of exogenous calmodulin in all four cases. The levels of 43S complexes were significantly elevated with all four antagonists, indicating a block in the utilization of 43S complexes. The similarity of the effects of four distinct classes of antagonists and their ready reversal by exogenous calmodulin leads us to suggest that there may be a role for calmodulin or a very similar calcium-binding protein in protein synthesis.

Regulation of the Ca(2+)-force relationship in permeabilized arterial smooth muscle.

It is generally accepted that the primary trigger for smooth muscle contraction is the elevation of intracellular Ca2+ concentration ([Ca2+]i) and subsequent phosphorylation of myosin light chain (MLC) by the Ca2+-calmodulin dependent MLC kinase (for reviews see Kamm and Stull, 1985; Somlyo, 1985). However, simultaneous measurements of tension and [Ca2+]i in intact tissues have shown that during continuous stimulation, although a-adrenergic agonist induced force is maintained at high constant levels, [Ca2+]i falls close to basal concentrations (Morgan and Morgan, 1982; 1984a). It has also been demonstrated that levels of both MLC phosphorylation and shortening velocity fall to suprabasal levels during the phase of force maintenance in intact smooth muscle (Dillon et al., 1981). In order to account for these phenomena, a high Ca2+ sensitivity state, the latch state, was proposed to be important for the maintenance of developed force in the face of significant decreases in the [Ca2+]i and levels of MLC phosphorylation (Dillon et al., 1981). Similar phenomena, increases in the Ca2+-force relationship, have been demonstrated in skinned smooth muscle preparations by the addition of exogenous calmodulin (Cassidy et al., 1981; Rüegg and Paul, 1982), an initial stimulation in high [Ca2+] followed by exposure to a lower [Ca2+] (Chatterjee and Murphy, 1981; Moreland and Murphy, 1986), and stimulation by phorbol esters (Chatterjee and Tajeda, 1986; Itoh et al., 1988) or GTPγS (Fujiwara et al., 1989). However, until recently it has not been possible to increase the Ca2+ sensitivity of force by a physiological mode of stimulation. Recent studies utilizing the Staphylcoccal α-toxin permeabilized smooth muscle preparation have shown that receptor stimulation by a physiological agonist plus GTP can produce a significant level of additional force, at a fixed submaximal [Ca2+], as compared to the force developed in response to Ca2+ alone (Nishimura et al., 1988; Kitazawa et al., 1989; Kobavashi et al., 1989).KeywordsMyosin Light ChainSmooth Muscle ContractionSmooth Muscle TissueForce RelationshipSmooth Muscle PreparationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.