Effects of trace amounts of Ca2+ and Mg2+ on the polymerization of actin

At low ionic strength, removal of trace amounts of Ca²⁺ enhanced the polymerization of actin, and that of Mg²⁺ inhibited it. The actions of the divalent metal ions did not influence the elongation process of preformed nuclei. Thus it appears that the metal ions affect the nucleation step of actin polymerization.     

Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca²⁺ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca²⁺-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca²⁺-ATPase activity was related to an increase in the apparent affinity for Ca²⁺ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca²⁺ homeostasis.     

Changes in conformation and nucleotide binding of Ca, Mn, or MgG-actin upon removal of the bound divalent cation. Studies of ultraviolet difference spectra and optical rotation

The ultraviolet difference spectra of EDTA-induced denaturation of dithiothreitoltreated actin prepared with either Ca²⁺, Mn²⁺, or Mg²⁺ as the strongly bound cation showed no appreciable difference, nor could any difference be found in the change of optical rotation. However, at different wavelengths the changes in the spectra have different rates and these rates do differ significantly depending on the bivalent cation bound to G-actin. The nucleotide and the cation appear to be removed simultaneously and at the fastest rate; about 50–80% is released within 1 min. The spectral changes have two phases: a fast change whose detailed kinetics have not been investigated in this paper, followed by a slower rate with first-order kinetics. The changes of optical rotation follow a single-phase first-order kinetics. The rates depend on the divalent cation, the sequence being Mn²⁺ > Ca²⁺ > Mg²⁺. ATP release is partially reversible upon Ca²⁺ addition; the reversibility is diminished as the time of incubation with EDTA is increased. On rebinding of ATP and Ca²⁺, the spectral and optical rotatory changes are not reversed, but no further changes occur. Such an EDTA-treated actin is polymerizable after addition of Ca²⁺, and the G-actin obtained after polymerization and depolymerization shows the same spectral change on a second addition of EDTA as the original actin. On the basis of these observations a scheme is suggested for the denaturation of G-actin.     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

Non-covalent binding of phosphate ions by striated muscle actin

Equilibrium dialysis experiments have shown that G-actin preparations can bind up to 9 phosphate ions and 13 vanadate ions per actin monomer with association constants of 3.00 × 10² M⁻¹ and 1.24 × 10² M⁻¹, respectively. Phosphate binding at low ionic strength caused removal of bound Ca²⁺ from G-actin and polymerization of the actin. The phosphate-treated polymeric actin was much more resistant to Pronase digestion than Ca²⁺- free polymeric action which did not contain bound phosphate but which was prepared by dialysis against EGTA-containing buffer. Vanadate-treated actin only polymerized to 47% of the extent of polymerization measured for phosphate-treated actin, indicating that vanadate ion is not as effective a promoter of low-ionic strength actin polymerization as phosphate ion.     

ATP und Ca2+ induzierte actomyosinartige Kontraktion glycerinisierter Makronuklei

Summary The addition of 2 mM-3 mM ATP to macronuclei ofParamecium bursaria suspended in a glycerol buffer medium causes a decrease in their volume up to 23% within 3 minutes. The infiltration medium must not only contain Ca²⁺, but must also be of low ionic strength for ATP to be effective. A slow, careful exchange of the glycerol medium for the contraction solution is also necessary. Ca²⁺ present alone in the standard contraction buffer can likewise induce a limited volume decrease; in the presence of low concentrations of Ca²⁺, Mg²⁺ shows no detectable effect on glycerinated nuclei. When the nuclear volume has been reduced by ATP in the presence of Ca²⁺, the addition of EGTA induces a reexpansion of the nuclei. Salyrgan, an organic mercurial, either prevents or abolishes the ATP-induced contraction. Other nucleotide triphosphates such as guanosine triphosphate (GTP), inosine triphosphate (ITP) or uridine triphosphate (UTP) likewise induce a volume decrease of the glycerinated macronuclei, but to a distinctly lesser extent than ATP.The results indicate that the volume decrease caused by the ATP-contraction solution is not a passive osmotic process. The resemblance to actomyosin contractions suggests that the volume decrease reported here might also be the result of the reaction of nuclear actomyosin and ATP.

     

Cytochalasins inhibit nuclei-induced actin polymerization by blocking filament elongation

Polylysine was found to induce polymerization of muscle actin in a low ionic strength buffer containing 0.4 mM MgCl2. The rate of induced polymerization was dependent on the amount and on the molecular size of the polylysine added. A similar effect was obtained by adding actin nuclei (containing about 2-4 actin subunits) cross-linked by p-N,N'- phenylenebismaleimide to G-actin under the same conditions, suggesting that the effect of polylysine is due to promotion of the formation of actin nuclei. Polymerization induced by polylysine and by cross-linked actin nuclei was inhibited by low concentrations (10(-8)-10(-6)M) of cytochalasins. Binding experiments showed that actin filaments, but not actin monomers, contained high-affinity binding sites for [3H]cytochalasin B (one site per 600 actin monomers). The relative affinity of several cytochalasins for these sites (determined by competitive displacement of [3H]dihydrocytochalasin B) was: cytochalasin D greater than cytochalasin E approximately equal to dihydrocytochalasin B. The results of this study suggest that cytochalasins inhibit nuclei-induced actin polymerization by binding to highly specific sites at the point of monomer addition, i.e., the elongation site, in actin nuclei and filaments.     

Interaction of anthracycline antibiotics with actin and heavy meromyosin.

For the purpose of elucidating the biochemical mechanism of anthracycline cardiomyopathy, the interaction with actin and heavy meromyosin (HMM) was studied. HMM and acto-HMM Mg²⁺-ATPase reactions were inhibited by daunorubicin and adriamycin; but not significantly by aclacinomycin A. The three antibiotics induced G-actin polymerization. Difference absorption spectra showed a direct interaction of adriamycin or aclacinomycin A with actin or HMM. Equilibrium dialysis and spectrofluorometric studies indicated that actin monomer possesses one binding site for adriamycin or aclacinomycin A with the same order of association constants (1.4 – 7.2 × 10⁴ M^?1). Adriamycin exhibited significantly higher affinity for HMM than aclacinomycin A.     

Tb3+ as a luminescent probe of actin structure: effects of polymerization, KI, and the binding of deoxyribonuclease I

Tb³⁺ can be introduced into the structure of G-actin by dialysis. Quantitation of the amounts of Tb³⁺ and Ca²⁺ bound to such actin samples reveals that, when Tb³⁺ is present in the dialysate at the same concentration as ATP, 0.26 mole of Tb³⁺ replaces an equivalent amount of Ca²⁺ per mole of G-actin. The resultant Tb³⁺ -containing actin solutions are nonturbid, undergo reversible salt-induced polymerization, and are indistinguishable from conventional G-actin preparations in their ability to inhibit the enzymatic activity of pancreatic deoxyribonuclease I. Illumination of the Tb³⁺ -containing actin solutions with 295 nm light produces, through an energy transfer process, an enhancement of Tb³⁺ emission intensities of some 100-fold over those for the protein-free, Tb³⁺ -containing dialysate. Polymerization of the G-actin in such samples by the addition of KCl or of MgCl₂ yields a further enhancement of about 1.5-fold in emission intensities. The responses to addition of KCl or of MgCl₂ differ quantitatively. The addition of KI to F-actin samples leads to a further enhancement in emission intensities, indicating that the structure of the actin monomers produced differs from that of either the G or F forms of actin unit. The binding of DNase I to Tb³⁺ -substituted G-actin results in a conformational change that is localized near to the site of binding of the Tb³⁺, as indicated by a net decrease in the measured intensity of emission.     

Binding stoichiometry of gadolinium to actin: its effect on the actin-bound divalent cation.

Using ⁴⁵Ca²⁺ and ¹⁵³Gd³⁺ we studied the effects of binding the lanthanide ion, gadolinium, to skeletal muscle G-actin. Gd³⁺ can specifically displace 6–7 Ca²⁺ from their binding sites on actin. Furthermore, a total of 6–7 Gd³⁺ can be shown to bind to actin, and this result is not affected by the subsequent addition of polymerizing quantities of KCl. We conclude that Gd³⁺ binds only to the Ca²⁺-binding sites of actin. The number of these Gd³⁺ sites closely corresponds to the known number of high and low affinity sites for divalent cations such as Ca²⁺.     

Similar affinities of ADP and ATP for G-actin at physiological salt concentrations

The equilibrium constant for the exchange of ATP and ADP at G-actin was determined by fluorimetric titration of G-actin-bound ε-ATP by ATP or ADP. The affinity of ATP for G-actin was found to be only about 3-fold higher than the affinity of ADP for G-actin at 37°C, pH 7.5 and physiologically relevant salt concentrations (100 mmol K⁺/l, 0.8 mmol Mg²⁺/l, <0.01 mmol Ca²⁺/l).     

Ionic Strength Dependence of Calcium, Adenine Nucleotide, Magnesium, and Caffeine Actions on Ryanodine Receptors in Rat Brain

Abstract: [³H]Ryanodine binding studies of ryanodine receptors in brain membrane preparations typically require the presence of high salt concentrations in assay incubations to yield optimal levels of binding. Here, radioligand binding measurements on rat cerebral cortical tissues were conducted under high (1.0 M KCI) and low (200 mM KCI) salt buffer conditions to determine the effects of ionic strength on receptor binding properties as well as on modulation of ligand binding by Ca²⁺, Mg²⁺, β,γ-methylene-adenosine 5′-triphosphate (AMP-PCP), and caffeine. In 1.0 M KCI buffer, labeled titration/equilibrium analyses yielded two classes of binding sites with apparent K_D (nM) and B_max (fmol/mg of protein) values of 2.4 and 34, respectively, for the high-affinity site and 19.9 and 157, respectively, for the low-affinity site. Unlabeled titration/equilibrium measurements gave a single high-affinity site with a K_D value of 1.9 nM and a B_max value of 95 fmol/mg of protein. The apparent K_D value derived from association and dissociation studies was 20 pM. Equilibrium binding was activated by Ca²⁺ (K_D/Ca²⁺= 14 nM), inhibited by Mg²⁺ (IC₆₀= 5.0 mM), and unaffected by AMP-PCP or caffeine. In 200 mM KCI buffer conditions, labeled titration analyses gave only a single site with a K_D value similar to and a B_max value 1.8-fold greater than those obtained for the low-affinity site in 1.0 M KCI buffer. In unlabeled titration measurements, the K_D value was fivefold lower, whereas the B_max value was unaffected. The K_D value derived from association and dissociation analysis was 2.4-fold greater in 200 mM KCI compared with 1.0 M KCI buffer conditions. In 200 mM compared with 1.0 M KCI, the potency with which Mg²⁺ inhibited binding was increased by 3.8-fold, whereas the affinity of the activation site for Ca²⁺ was reduced by 13-fold. Addition of caffeine in the presence of low salt increased the affinity of Ca²⁺ activation by 1.7-fold. The inhibitory effect of Mg²⁺ on [³H]-ryanodine binding in the presence of 200 mM KCI was reversed by AMP-PCP and caffeine with apparent EC₅₀ values of 0.25 and 7.6 mM, respectively. Taken together, these results indicate that ionic strength is an important consideration in binding studies of brain ryanodine receptors and their interactions with modulatory agents.     

Calcium binding to rabbit skeletal myosin under physiological conditions

At a free Mg²⁺ concentration of 1.0 mM, myosin binds one Ca²⁺ per molecule when the Ca²⁺ concentration is 20 μM, a value in the concentration range expected during contraction of skeletal muscle. Mg²⁺ alters Ca²⁺ binding in a complex manner, not by simple competition. In the range from 20 to 100 μM Mg²⁺ it produces positive cooperativity between the high-affinity Ca²⁺ binding sites, in addition to shifting binding to higher Ca²⁺ concentrations. High-affinity Ca²⁺ binding is not significantly affected by the addition of ATP, increase in ionic strength to 0.1 and changes in temperature. Ca²⁺ binding did not increase actin-activated ATPase activity in the absence of regulatory proteins, but rather inhibited it.     

Compressibility and specific volume of actin decrease upon G to F transformation

We measured the densities as well as the sound velocities in solutions of G-actin, F-actin and the reconstituted thin filament. Using the data obtained, we determined their partial specific volumes and partial specific adiabatic compressibilities. The objectives were to investigate the volume change of actin upon polymerization and to detect the conformational change associated with the Ca²⁺-binding to the reconstituted thin filament. The partial specific volume and the partial specific adiabatic compressibility of G-actin were 0.749 cm³/g and 9.3 · 10⁻¹² cm²/dyne, respectively. The results suggest that G-actin is a rather soft protein compared with other globular proteins. The partial specific volumes of F-actin were in a range of 0.63–0.66 cm³/g depending on the solvent conditions. The partial specific adiabatic compressibilities of F-actin were negative (−(7–13) · 10⁻¹² cm³/dyne). These data indicate that the amount of hydration may increase by several times upon polymerization assuming that the size of the cavity remains constant. We detected little difference between the partial specific adiabatic compressibility of the reconstituted thin filament in a Ca²⁺-bound state and that in a Ca²⁺-unbound state. This suggests that the Ca²⁺ binding affected not the subunit itself but the inter-subunit junction.     

Actin and Myosin in pea tendrils

We demonstrate here the presence of actin and myosin in pea (Pisum sativum L.) tendrils. The molecular weight of tendril actin is 43,000, the same as rabbit skeletal muscle actin. The native molecular weight of tendril myosin is about 440,000. Tendril myosin is composed of two heavy chains of molecular weight approximately 165,000 and four (two pairs) light chains of 17,000 and 15,000. At high ionic strength, the ATPase activity of pea tendril myosin is activated by K⁺-EDTA and Ca²⁺ and is inhibited by Mg²⁺. At low ionic strength, the Mg²⁺-ATPase activity of pea tendril myosin is activated by rabbit skeletal muscle F-actin. Superprecipitation occurred after incubation at room temperature when ATP was added to the crude actomyosin extract. It is suggested that the interaction of actin and myosin may play a role in the coiling movement of pea tendril.     

Intramolecular domain-domain interactions and intermolecular self-association in bovine prothrombin. A potentiometric and laser light-scattering study

The interaction of bovine prothrombin with Ca²⁺ and Mg²⁺ ions was investigated by following H⁺ release as a function of metal ion concentration at pH 6 and pH 7.4 at high and low ionic strength. Prothrombin Ca²⁺ and Mg²⁺ binding is characterized by high- and low-affinity sites. M²⁺ binding at these sites is associated with intramolecular conformational changes and also with intermolecular self-association. The pH dependence of H⁺ release by M²⁺ is bell shaped and consistent with controlling pK_a values of 4.8 and 6.5. At pH 6 and low ionic strength, both Ca²⁺ and Mg²⁺ titrations following H⁺ release clearly show independent low- and high-affinity binding sites. Laser light scattering reveals that at pH 7.4 and low ionic strength, and at pH 6.0 and high ionic strength, the prothrombin molecular weight is between 73 and 98 kD. At pH 7.4 and high ionic strength, prothrombin is monomeric in the absence of metal ions, but appears to dimerize in the presence of M²⁺. At pH 6.0 and low ionic strength prothrombin exists as a dimer in the absence of metal ions and is tetrameric in the presence of Ca²⁺ and remains dimeric in the presence of Mg²⁺. These results and those for metal ion-dependent H⁺ release indicate that H⁺ release occurs concomitantly with association processes involving prothrombin.Abbreviations GLA -carboxyglutamic acid; fragment 1. amino terminal residues 1–156 of bovine prothrombin - MES 2-(N-morpholino) ethanesulfonic acid - MOPS 3-(N-morpholino) propanesulfonic acid - PS/PC phosphatidylserine/phosphatidylcholine vesicles - ionic strength     

Rabbit skeletal muscle F-actin can be stable at low ionic strength, provided trace amounts of Ca2+ are absent.

Addition of low concentrations (0.2--2.0 mM) of EGTA to rabbit skeletal muscle G-actin in the presence of ATP caused increase in viscosity. The effect is probably due to chelation of Ca2+. EGTA-polymerized actin was sedimented in the ultracentrifuge as a pellet which could be depolymerized in the presence of Ca2+ and then repolymerized. Electron microscopy indicated that formation of filamentous actin which appears to be somewhat more flexible than F-actin obtained by polymerization with KCl. The EGTA-polymerized actin was dissociated by DNAase I faster than KCl-polymerized actin. F-Actin can thus be stable also in very low ionic strength media if Ca2+ is removed whereas for G-actin to be the only form of the protein in such media, micromolar concentrations of Ca2+ must be present.     

Control of insulin receptor affinity by a Ca2+-sensitive binding site

Calcium (Ca²⁺) increased insulin-receptor binding in both membrane and solubilised receptor preparations. Ca²⁺ increased both receptor affinity and initial rate of association of [¹²⁵I]insulin to the receptor preparations. Ca²⁺ had no effect on insulin receptor number in either receptor preparation. The effect of Ca²⁺ on affinity could be mimicked by ions with similar ionic radii and properties (e.g., Ba²⁺, Mg²⁺ and Sr²⁺). EDTA and oleic acid reduced insulin binding and receptor affinity and these effects were reversed by the addition of Ca²⁺. These studies suggest that Ca²⁺ and Ca²⁺-like ions may bind to a site on or near the receptor and may be responsible for a conformational change with a consequent increase in receptor affinity.     

Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum

A myosin-like protein was purified from amoebae of the cellular slime mold Dictyostelium discoideum. The purification utilized newly discovered solubility properties of actomyosin in sucrose. The amoebae were extracted with a 30% sucrose solution containing 0.1 m-KCl, and actomyosin was selectively precipitated from this crude extract by removal of the sucrose. The myosin and actin were then solubilized in a buffer containing KI and separated by gel filtration.The purified Dictyostelium myosin bears a very close resemblance to muscle myosin. The amoeba protein contains two heavy chains, about 210,000 molecular weight each, and two classes of light chains, 16,000 and 18,000 molecular weight. Dictyostelium myosin is insoluble at low ionic strength and forms bipolar thick filaments. The myosin possesses ATPase activity that is activated by Ca²⁺ but not EDTA, and is inhibited by Mg²⁺; under optimal conditions the specific activity of the enzyme is 0.09 μmol P₁/min per mg myosin.Dictyostelium myosin interacts with Dictyostelium actin or muscle actin, as shown by electron microscopy and by measurements of enzymatic activity. The ATPase activity of Dictyostelium myosin, in the presence of Mg²⁺ at low ionic strength, exhibits an average ninefold activation when actin is added.     

The mechanochemical basis of amoeboid movement: II. Cytoplasmic filament stability at low divalent cation concentrations

The role played by Ca²⁺ in the stability of cytoplasmic actin and myosin filaments was investigated ultrastructurally with negatively stained isolated cytoplasm from Chaos carolinensis. Cytoplasm was incubated in solutions containing 5, 10, 15 and 25 mM EGTA for periods of time varying from 2 to 20 min. As either the EGTA concentration or duration of incubation was increased, the extent of myosin and actin filament depolymerization increased. The actin filaments depolymerized except where they were stabilized by interaction with myosin. With longer incubation times or higher EGTA concentrations complete depolymerization of the actin filaments could be accomplished. Myosin aggregates also disassembled and became shorter, while monomeric myosin labelled adjacent thin filaments to form arrowhead complexes resembling myosin enriched actomyosin [1]. These actomyosin complexes were relatively stable at low Ca²⁺ concentrations. In addition, the complexes showed a characteristic 35 nm periodicity and were dissociable in the presence of Mg²⁺-ATP. The actin containing filaments were more labile at low Ca²⁺ concentrations than the myosin aggregates. These results suggest that in cells capable of regulating their Ca²⁺ concentrations efficiently, filament polymerization-depolymerization could play a role in the control of cytoplasmic streaming.