Actin filament capping protein from bovine brain.

An actin filament capping protein has been purified from bovine brain. The protein has a native mol. wt. of 63 kilodaltons (kd) with subunits of 36 kd and 31 kd and is globular in shape. It nucleates actin polymerization, inhibits filament elongation and filament interactions, and decreases the steady state viscosity of F-actin in substoichiometric amounts (molar ration 1:1000). In addition, the protein increases the critical concentration for actin polymerization. Neither Ca2+ nor calmodulin affects it action. All these effects can be explained by the binding of the protein to the 'barbed' end of actin filaments leading to a blockade of actin monomer addition at the preferred growing end. This is directly demonstrated by electron microscopy. Concerning the polypeptide composition, Ca2+-independence, mode, and stoichiometry of actin interaction, the protein is similar to the capping protein, previously isolated from Acanthamoeba.     

A 90 000-dalton actin-binding protein from platelets. Comparison with villin and plasma brevin

Affinity chromatography of Ca2+-containing extracts of platelets on DNAase I-Sepharose, using Ca2+-free buffer as eluant, selects a 1:1 complex of a 90 000-dalton protein with actin. The complex shows little interaction with either DNAase or actin unless Ca2+ is present. In the presence of Ca2+, the complex nucleates polymerization of actin, reduces the viscosity attained, and delays filament formation from profilactin with characteristics closely resembling those shown by chicken villin. Proteolysis of the native proteins indicates structural similarity between the platelet protein and villin or villin core; limited proteolytic digestion in the presence of SDS distinguishes the platelet protein from villin but not from the functionally related plasma protein, brevin. The platelet protein is not accessible to enzyme-mediated iodination of surface components on intact cells. The term 'platelet brevin' is proposed for the protein.     

Interaction of actinogelin with actin. No nucleation but high gelation activity

Nucleation activity of actin polymerization of actinogelin, a calcium-sensitive F-actin cross-linking protein from rat liver, was measured by a fluorescence enhancement method using pyrenyl-actin and by high shear viscometry. No stimulation of nucleation by the addition of actinogelin was observed under several ionic conditions using the fluorescent method. Similar results were also obtained by viscometry. Therefore, it can be concluded that actinogelin has no nucleation activity for actin polymerization. By electron microscopy, it was found that actinogelin molecule has a dumbbell shape, binds to side of F-actin through its end(s), and cross-links actin filaments by binding with its two ends. It was also found that meshwork formation occurred in low Ca2+ conditions from F-actin and actinogelin. Under non-gelling high Ca2+ conditions, binding of actinogelin along the side of F-actin with its one end was still detected in accordance with the binding assay using ultracentrifugation and protein determination. Under low Ca2+ conditions, the critical gelling concentration of actinogelin measured by low shear viscometry at 20 degrees C was 6 micrograms/ml for 250 micrograms/ml of actin. Comparing this value with those of the other actin cross-linking proteins, it was found that actinogelin was one of proteins with the highest gelation activity. On the other hand, gelation activity of actinogelin in high Ca2+ conditions was one order of magnitude lower; more than 50 micrograms/ml of the protein was required for gelation. At 37 degrees C, gelation activity of actinogelin at low Ca2+ concentration was decreased to about a quarter of that at 20 degrees C, but this was still higher than that of gizzard alpha-actinin at 20 degrees C. Thus, role of actinogelin as an efficient and Ca2+-regulated cross-linker of microfilaments was substantiated.     

Isolation of calcium-dependent platelet proteins that interact with actin

Low Ca2+ extracts of platelets rapidly form an actin gel when warmed to 25 degrees C. The addition of Ca2+ has three effects. At Ca/EGTA = 0.4, the gel begins to contract. Increasing the Ca2+ concentration increases the rate of contraction and reduces the amount of actomyosin gel. Between Ca/EGTA = 0.4 and 0.5, a protease is activated that selectively degrades polypeptides with molecular weight greater than the myosin heavy chain. At Ca/EGTA = 1, about 70% of the total actin is nonsedimentable. Addition of excess EGTA produces the rapid formation of an actomyosin gel, which is not readily solubilized by re-addition of calcium. Using DNAase l-Sepharose chromatography, we have isolated a protein fraction whose binding to actin is Ca2+ -dependent. This fraction contains a major polypeptide with a molecular weight of 90,000. This fraction increases the rate of development of high sheer viscosity, but lowers the final value if Ca2+ is present. This decrease in viscosity is due to the generation of shorter filaments. In the presence of Ca2+, this protein(s) selectively blocks the addition of actin monomers to the barbed end of glutaraldehyde-fixed S1-decorated actin fragments and will nucleate assembly of filaments. We speculate that this protein(s) may serve as a Ca2+ -dependent nucleation site in situ.     

Actin polymerization in neutrophils is triggered without a requirement for a rise in cytoplasmic Ca2+.

Stimulation of rat neutrophils with the peptide fMetLeuPhe caused (i) the appearance of a 40 kDa protein in the Triton-X-100-insoluble cytoskeleton, (ii) the disappearance of DNAase inhibition from the cytosol and (iii) the appearance of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phallacidin (NBD-phallacidin) binding sites. All three observations were consistent with a rapid and transient assembly of polymerized actin, peaking at approximately 5 s and returning to near resting levels within 40 s. By experimentally depleting the cells of Ca2+ and increasing the cytoplasmic Ca2+ buffering capacity, the peptide-induced Ca2+ transient was reduced from a peak of 900 nM to 250 nM, without inhibiting actin polymerization, and this peak was sustained for at least 2 min. A further dissociation between the triggering of actin polymerization and peptide-induced Ca2+ elevation and oxidase activation was demonstrated at low concentrations of peptide (1-100 pM), actin polymerization being triggered without an elevation in Ca2+ or activation of the oxidase. Two other agents which induced actin polymerization, phorbol 12-myristate 13-acetate and latex beads, failed to elevate cytoplasmic Ca2+. It was therefore concluded that neither Ca2+ nor those intracellular messengers which act with Ca2+ to trigger the neutrophil oxidase are responsible for triggering actin polymerization in neutrophils.     

Ca2+ regulation of gelsolin activity: binding and severing of F-actin.

Regulation of the F-actin severing activity of gelsolin by Ca2+ has been investigated under physiologic ionic conditions. Tryptophan fluorescence intensity measurements indicate that gelsolin contains at least two Ca2+ binding sites with affinities of 2.5 x 10(7) M-1 and 1.5 x 10(5) M-1. At F-actin and gelsolin concentrations in the range of those found intracellularly, gelsolin is able to bind F-actin with half-maximum binding at 0.14 microM free Ca2+ concentration. Steady-state measurements of gelsolin-induced actin depolymerization suggest that half-maximum depolymerization occurs at approximately 0.4 microM free Ca2+ concentration. Dynamic light scattering measurements of the translational diffusion coefficient for actin filaments and nucleated polymerization assays for number concentration of actin filaments both indicate that severing of F-actin occurs slowly at micromolar free Ca2+ concentrations. The data suggest that binding of Ca2+ to the gelsolin-F-actin complex is the rate-limiting step for F-actin severing by gelsolin; this Ca2+ binding event is a committed step that results in a Ca2+ ion bound at a high-affinity, EGTA-resistant site. The very high affinity of gelsolin for the barbed end of an actin filament drives the binding reaction equilibrium toward completion under conditions where the reaction rate is slow.     

Mechanism of CaCl2-induced actin polymerization

The CaCl2 concentration dependence of the rate of actin filament elongation and of the actin monomer concentration at steady state with actin polymer (the critical actin concentration) has been investigated. A relative rate of actin filament elongation from actin polymer intermolecularly cross-linked with N,N'-p-phenylenebis(maleimide) showed a sigmoidal dependence on the concentration of CaCl2 used to induce actin polymerization. This result is shown to be consistent with a model in which only actin monomer containing five equivalently bound Ca2+ ions (Ka = 2 mM-1) is capable of addition to actin polymer. A relative dissociation rate constant for actin monomer removal from polymer was calculated from the product of the critical actin concentration and the relative elongation rate constant and was found to be virtually independent of CaCl2 concentration. The relationship between Ca2+ binding sites on actin and the CaCl2 concentration dependence of the kinetics of actin filament elongation is discussed.     

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.     

Gelsolin as a calcium-regulated actin filament-capping protein.

Various concentrations of gelsolin (25-100 nM) were added to 2 microM polymerized actin. The concentrations of free calcium were adjusted to 0.05-1.5 microM by EGTA/Ca2+ buffer. Following addition of gelsolin actin depolymerization was observed that was caused by dissociation of actin subunits from the pointed ends of treadmilling actin filaments and inhibition by gelsolin of polymerization at barbed ends. The time course of depolymerization revealed an initial lag phase that was followed by slow decrease of the concentration of polymeric actin to reach the final steady state polymer and monomer concentration. The initial lag phase was pronounced at low free calcium and low gelsolin concentrations. On the basis of quantitative analysis the kinetics of depolymerization could be interpreted as capping, i.e. binding of gelsolin to the barbed ends of actin filaments and subsequent inhibition of polymerization, rather than severing. The main argument for this conclusion was that even gelsolin concentrations (100 nM) that exceed the concentration of filament ends ( approximately 2 nM), cause the filaments to depolymerize at a rate that is similar to the rate of depolymerization of the concentration of pointed ends existing before addition of gelsolin. The rate of capping is directly proportional to the free calcium concentration. These experiments demonstrate that at micromolar and submicromolar free calcium concentrations gelsolin acts as a calcium-regulated capping protein but not as an actin filament severing protein, and that the calcium binding sites of gelsolin which regulate the various functions of gelsolin (capping, severing and monomer binding), differ in their calcium affinity.     

Effect of pH on the mechanism of actin polymerization

The effect of pH on the Mg2+-induced polymerization of rabbit skeletal muscle G-actin at 20 degrees C was examined. Polymerization data were obtained at various initial concentrations of Mg2+, Ca2+, and G-actin between pH 6 and 7.5. The data were found to fit a kinetic mechanism for actin polymerization previously proposed at pH 8 in which Mg2+ binding at a moderate-affinity site on actin induces an isomerization of the protein enabling more favorable nucleation [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886]. The data also suggest the formation of actin dimers induced by Mg2+ binding is over 2 orders of magnitude more favorable at pH 6 than at pH 8. Little effect on trimer formation is found over this pH range. In addition, the conformation induced by nonspecific binding of metal to low-affinity sites becomes more favorable as the pH is lowered. The critical concentration for filament formation is also decreased at lower pH. The kinetic data do not support fragmentation occurring under any of the conditions examined. Furthermore, as Mg2+ exchange for Ca2+ at a high-affinity site (Kd less than 10(-9) M) fails to alter significantly the polymerization kinetics, Ca2+ release from this site appears unnecessary for either the nucleation or the elongation of actin filaments.     

Purification and characterization of a Ca2+-dependent actin filament severing protein from bovine adrenal medulla

We describe the purification of an actin regulatory protein from bovine adrenal medulla. This protein caused a dose-dependent decrease of the specific viscosity of actin solution within 30 s of its addition in a Ca2+-sensitive way. Sedimentation assays and the observation by electron microscopy showed that this effect was ascribable to the fragmentation of actin filaments. This protein apparently promoted nucleation of actin polymerization and increased the critical concentration of actin for polymerization nearly 5-fold, suggesting its binding to the barbed end of actin filaments. The inhibitory effect of this protein on the elongation of actin from the barbed end of the myosin subfragment S1-labeled actin seeds confirmed this suggestion. These properties are similar to those of gelsolin. However, the physicochemical properties of this protein having a single polypeptide chain with a molecular weight of 74,000, a Stokes radius of 3.9 nm, a sedimentation coefficient (s0(20),w) of 4.5 S, and an immunological characterization showed that this protein is different from gelsolin.     

Regulation of calcium-dependent [3H]noradrenaline release from rat cerebrocortical synaptosomes by protein kinase C and modulation of the actin cytoskeleton.

The effects that active phorbol esters, staurosporine, and changes in actin dynamics, might have on Ca2+ -dependent exocytosis of [3H]-labelled noradrenaline, induced by either membrane-depolarizing agents or a Ca2+ ionophore, have been examined in isolated nerve terminals in vitro. Depolarization-induced openings of voltage-dependent Ca2+ channels with 30 mM KCl or 1 mM 4-aminopyridine induced limited exocytosis of [3H]noradrenaline, presumably from a readily releasable vesicle pool. Application of the Ca2+ ionophore calcimycin (10 microM) induced more extensive [3H]noradrenaline release, presumably from intracellular reserve vesicles. Stimulation of protein kinase C with phorbol 12-myristate,13-acetate increased release evoked by all secretagogues. Staurosporine (1 microM) had no effect on depolarization-induced release, but decreased ionophore-induced release and reversed all effects of the phorbol ester. When release was induced by depolarization, internalization of the actin-destabilizing agent DNAase I into the synaptosomes gave a slight increase in [3H]NA release and strongly increased the potentiating effect of the phorbol ester. In contrast, when release was induced by the Ca2+ ionophore, DNAase I had no effect, either in the absence or presence of phorbol ester. The results indicate that depolarization of noradrenergic rat synaptosomes induces Ca2+ -dependent release from a releasable pool of staurosporine-insensitive vesicles. Activation of protein kinase C increases this release by staurosporine-sensitive mechanisms, and destabilization of the actin cytoskeleton further increases this effect of protein kinase C. In contrast, ionophore-induced noradrenaline release originates from a pool of staurosporine-sensitive vesicles, and although activation of protein kinase C increases release from this pool, DNAase I has no effect and also does not change the effect of protein kinase C. The results support the existence of two functionally distinct pools of secretory vesicles in noradrenergic CNS nerve terminals, which are regulated in distinct ways by protein kinase C and the actin cytoskeleton.     

An 100-kDa Ca2+-sensitive actin-fragmenting protein from unfertilized sea urchin egg

An actin-modulating protein was purified from unfertilized eggs of sea urchin, Hemicentrotus pulcherrimus, by means of DNase I affinity and DEAE-cellulose column chromatographies. This protein was a globular protein with a Stokes radius of 41-42 nm and consisted of a single polypeptide chain having an apparent molecular mass of 100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Gel filtration chromatography revealed that one 100-kDa protein molecule binds two or three actin monomers in the presence of Ca2+, but such binding was not observed in the absence of Ca2+. The effect of the 100-kDa protein on the polymerization of actin was studied by viscometry, spectrophotometry and electron microscopy. The initial rate of actin polymerization was decreased at a very low molar ratio of 100-kDa protein/actin. Acceleration of the initial rate of polymerization occurred at a relatively high, but still substoichiometric, molar ratio of 100-kDa protein/actin. The 100-kDa protein produced fragmentation of muscle actin filaments at Ca2+ concentrations greater than 0.3 microM as revealed by viscometry and electron microscopy. Evidence was also presented that the 100-kDa protein binds to the barbed end of the actin filament.     

A 45,000-mol-wt protein from unfertilized sea urchin eggs severs actin filaments in a calcium-dependent manner and increases the steady-state concentration of nonfilamentous actin

A 45,000-mol-wt protein has been purified from unfertilized sea urchin (Strongylocentrotus purpuratus) eggs. The isolation scheme includes DEAE cellulose ion-exchange chromatography, gel filtration, and hydroxylapatite chromatography. The homogeneity of the isolated protein is greater than 90% by SDS PAGE. The 45,000-mol-wt protein reduces the viscosity of actin filaments in a Ca2+-dependent manner. The free calcium concentration required for the activity of this protein is in the micromolar range. Electron microscopic studies reveal that the formation of short filaments parallels the decrease in viscosity. Energy transfer and sedimentation experiments indicate a net disassembly of actin filaments and an increase in the steady-state nonfilamentous actin concentration in the presence of Ca2+ ions and the 45,000-mol-wt protein. The increase in the steady-state nonfilamentous actin concentration is proportional to the amount of 45,000-mol-wt protein added. The actin molecules disassembled by the addition of the 45,000-mol-wt protein are capable of polymerization.     

Cap100, a novel phosphatidylinositol 4,5-bisphosphate-regulated protein that caps actin filaments but does not nucleate actin assembly.

The fast and transient polymerization of actin in nonmuscle cells after stimulation with chemoattractants requires strong nucleation activities but also components that inhibit this process in resting cells. In this paper, we describe the purification and characterization of a new actin-binding protein from Dictyostelium discoideum that exhibited strong F-actin capping activity but did not nucleate actin assembly independently of the Ca2+ concentration. These properties led at physiological salt conditions to an inhibition of actin polymerization at a molar ratio of capping protein to actin below 1:1,000. The protein is a monomer, with a molecular mass of approximately 100 kDa, and is present in growing and in developing amoebae. Based on its F-actin capping function and its apparent molecular weight, we designated this monomeric protein cap100. As shown by dilution-induced depolymerization and by elongation assays, cap100 capped the barbed ends of actin filaments and did not sever F-actin. In agreement with its capping activity, cap100 increased the critical concentration for actin polymerization. In excitation or emission scans of pyrene-labeled G-actin, the fluorescence was increased in the presence of cap100. This suggests a G-actin binding activity for cap100. The capping activity could be completely inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), and bound cap100 could be removed by PIP2. The inhibition by phosphatidylinositol and the Ca(2+)-independent down-regulation of spontaneous actin polymerization indicate that cap100 plays a role in balancing the G- and F-actin pools of a resting cell. In the cytoplasm, the equilibrium would be shifted towards G-actin, but, below the membrane where F-actin is required, this activity would be inhibited by PIP2.     

Calcium control of Saccharomyces cerevisiae actin assembly.

Low levels of Ca2+ dramatically influence the polymerization of Saccharomyces cerevisiae actin in KCl. The apparent critical concentration for polymerization (C infinity) increases eightfold in the presence of 0.1 mM Ca2+. This effect is rapidly reversed by the addition of ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid or of 0.1 mM Mg2+. Furthermore, the addition of Ca2+ to polymerized actin causes a reversible increase in the apparent C infinity. In the presence of Ca2+, at actin concentrations below the apparent C infinity, particles of 15 to 50 nm in diameter are seen instead of filaments. These particles are separated from soluble actin when Ca2+-treated filamentous actin is sedimented at high speed; both the soluble and particulate fractions retain Ca2+-sensitive polymerization. The Ca2+ effect is S. cerevisiae actin-specific: the C infinity for rabbit muscle actin is not affected by the presence of Ca2+ and S. cerevisiae actin. Ca2+ may act directly on S. cerevisiae actin to control the assembly state in vivo.     

Study of a protein complex from the brain which reduces actin viscosity. II. The complex inhibits elongation of actin filaments at the end preferable for polymerization and fragments the filaments

Functional properties of the protein complex from bovine brain that shortens actin filaments are described. In the presence of Ca2+ complex shortens actin filaments and increases the initial rate of actin polymerization. In the absence of free calcium ions the complex loses its accelerating effect on actin polymerization, but still possesses actin filament shortening activity. Neither phalloidin nor tropomyosin prevent the shortening of actin filaments induced by the protein complex. Therefore the protein complex causes the fragmentation of actin filament. The data on actin polymerization in the presence of F-actin nuclei have indicated that the protein complex inhibits the elongation step of actin polymerization. The analysis of elongation in the presence of both the protein complex and cytochalasin D has demonstrated that the inhibition occurs on the fast-growing end of actin filaments.     

Isolation and properties of two actin-binding domains in gelsolin

Gelsolin is a Ca2+-sensitive 90-kDa protein which regulates actin filament length. A molecular variant of gelsolin is present in plasma as a 93-kDa protein. Functional studies have shown that gelsolin contains two actin-binding sites which are distinct in that after Ca2+-mediated binding, removal of free Ca2+ releases actin from one site but not from the other. We have partially cleaved human plasma gelsolin with alpha-chymotrypsin and identified two distinct actin-binding domains. Peptides CT17 and CT15, which contain one of the actin-binding domains, bind to actin independently of Ca2+; peptides CT54 and CT47, which contain the other domain, bind to actin reversibly in response to changes in Ca2+ concentration. These peptides sequester actin monomers inhibiting polymerization. Unlike intact gelsolin, neither group of peptides nucleates actin assembly or forms stable filament end caps. CT17 and CT15 can however sever actin filaments. Amino acid sequence analyses place CT17 at the NH2 terminus of gelsolin and CT47 at the carboxyl-terminal two-thirds of gelsolin. Circular dichroism measurements show that Ca2+ induces an increase in the alpha-helical content of CT47. These studies provide a structural basis for understanding the interaction of gelsolin with actin and allow comparison with other Ca2+-dependent actin filament severing proteins.     

Mutational analysis of Ser14 and Asp157 in the nucleotide-binding site of beta-actin.

This paper compares wild-type and two mutant beta-actins, one in which Ser14 was replaced by a cysteine, and a second in which both Ser14 and Asp157 were exchanged (Ser14-->Cys and Ser14-->Cys, Asp157-->Ala, respectively). Both of these residues are part of invariant sequences in the loops, which bind the ATP phosphates, in the interdomain cleft of actin. The increased nucleotide exchange rate, and the decreased thermal stability and affinity for DNase I seen with the mutant actins indicated that the mutations disturbed the interdomain coupling. Despite this, the two mutant actins retained their ATPase activity. In fact, the mutated actins expressed a significant ATPase activity even in the presence of Ca2+ ions, conditions under which actin normally has a very low ATPase activity. In the presence of Mg2+ ions, the ATPase activity of actin was decreased slightly by the mutations. The mutant actins polymerized as the wild-type protein in the presence of Mg2+ ions, but slower than the wild-type in a K+/Ca2+ milieu. Profilin affected the lag phases and elongation rates during polymerization of the mutant and wild-type actins to the same extent, whereas at steady-state, the concentration of unpolymerized mutant actin appeared to be elevated. Decoration of mutant actin filaments with myosin subfragment 1 appeared to be normal, as did their movement in the low-load motility assay system. Our results show that Ser14 and Asp157 are key residues for interdomain communication, and that hydroxyl and carboxyl groups in positions 14 and 157, respectively, are not necessary for ATP hydrolysis in actin.     

The effects of Mg2+ at the high-affinity and low-affinity sites on the polymerization of actin and associated ATP hydrolysis

Actin contains a single high-affinity cation-binding site, for which Ca2+ and Mg2+ can compete, and multiple low-affinity cation-binding sites, which can bind Ca2+, Mg2+, or K+. Binding of cations to the low-affinity sites causes polymerization of monomeric actin with either Ca2+ or Mg2+ at the high-affinity site. A rapid conformational change occurs upon binding of cations to the low-affinity sites (G----G) which is apparently associated with the initiation of polymerization. A much slower conformational change (G----G', or G----G' if the low-affinity sites are also occupied) follows the replacement of Ca2+ by Mg2+ at the high-affinity site. This slow conformational change is reflected in a 13% increase in the fluorescence of G-actin labeled with the fluorophore 7-chloro-4-nitrobenzene-2-oxadiazole (NBD-labeled actin). The rate of the ATP hydrolysis that accompanies elongation is slower with Ca-G-actin than with Mg-G'-actin (i.e. with Ca2+ rather than Mg2+ at the high-affinity site) although their rates of elongation are similar. The slow ATP hydrolysis on Ca-F-actin causes a lag in the increase in fluorescence associated with the elongation of actin labeled with the fluorophore N-pyrene iodoacetamide (pyrenyl-labeled actin), even though there is no lag in the elongation rate, because pyrenyl-labeled ATP-F-actin subunits have a lower fluorescence intensity than pyrenyl-labeled ADP-F-actin subunits. The effects of the cation bound to the high-affinity binding site must, therefore, be considered in quantitatively analyzing the kinetics of polymerization of NBD-labeled actin and pyrenyl-labeled actin. Although their elongation rates are not very different, the rate of nucleation is much slower for Ca-G-actin than for Mg-G'-actin, probably because of the slower rate of ATP hydrolysis when Ca2+ is bound to the high-affinity site.