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.     

F-actin bundling protein from Physarum polycephalum: purification and its capacity for co-bundling of actin filaments and microtubules

An F-actin bundling protein was isolated and purified from plasmodium of Physarum polycephalum. The F-actin bundling protein in Physarum extract was passed through a DEAE-cellulose column. After the protein in the fraction was treated with 6 M urea, it was purified by gel filtration on Sephacryl S-300 HR followed by chromatography on CM-Toyopearl (cation exchange) in the presence of 6 M urea. The purified protein gave a single band on SDS-PAGE, and the molecular weight was estimated to be 52,000. This F-actin bundling protein is referred to as the 52 kDa protein. Interestingly, the 52 kDa protein also induced bundling of microtubules. The formation of F-actin and microtubule bundles was Ca(2+)-insensitive, but depended on the salt concentration. Each bundle formed at NaCl concentrations less than 0.1 M. The 52 kDa protein cross-reacted with monoclonal antibody raised against a HeLa 55 kDa protein (an F-actin bundling protein from HeLa cells) (Yamashiro-Matsumura and Matsumura: J. Biol. Chem. 260:5087-5097, 1985). When the 52 kDa protein was added to a mixture of actin filaments and microtubules, co-bundles composed of both filaments formed. This is the first reported example in which an F-actin bundling protein induced co-bundling of actin filaments and microtubules.     

An 18K protein from ascites hepatoma cell depolymerizes actin filaments rapidly

Several actin binding proteins were isolated from ascites hepatoma cells AH7974 by DNase I affinity chromatography. Among them, a protein having a molecular weight of 18,000 was further purified by DEAE cellulose and hydroxyapatite column chromatographies and gel filtration on a Sephadex G-75 column. The 18K protein not only inhibits actin polymerization but also depolymerizes actin filaments. This conclusion was supported by viscosity and fluorescence intensity measurements and the DNase I inhibition assay. A chemical cross-linking experiment suggested that the 18K protein binds to monomeric actin and forms and 18K-actin 1:1 complex. The net depolymerization rate by the 18K protein measured by the DNase I inhibition assay was slower than the rapid reduction of the fluorescence intensity of pyrene-labeled F-actin upon addition of the 18K protein. This result suggests that the 18K protein not only binds to monomeric actin but also binds to actin filaments directly. The sedimentation assay showed that a part of the 18K protein was cosedimented with actin filaments. Electron microscopic observations demonstrated that the 18K protein decreased the amount of actin filaments and the remaining filaments appeared to be decorated and distorted by the 18K protein. The 18K protein had no Ca2+ ion sensitivity and exhibited the same effect on both this tumor actin and muscle actin.     

Caldesmon: a common actin-linked regulatory protein in the smooth muscle and nonmuscle contractile system

Caldesmon was originally purified from gizzard smooth muscle as a major calmodulin-binding protein which also interacts with actin filaments. It has an alternative binding ability to either calmodulin or actin filaments depending upon the concentration of Ca2+ ("flip-flop binding"). Two forms of caldesmon (Mr's in the range of 120-150 kDa and 70-80 kDa) have been demonstrated in a wide variety of smooth muscles and nonmuscle cells. Immunohistochemical studies suggest that caldesmon is colocalized with actin filaments in vivo. Considering its abundance, the Ca2+-dependent flip-flop binding ability to either calmodulin or actin filaments, and its intracellular localization, caldesmon is expected to be involved in contractile events. Recent results from our laboratory have led to the conclusion that caldesmon regulates the smooth muscle and nonmuscle actin-myosin interaction and the smooth muscle actin-high Mr actin-binding protein (ABP or filamin) interactin in a flip-flop manner. It might function in cell motility by regulating the contractile system.     

The N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin complex

Apoptosis is essential in embryonic development, clonal selection of cells of the immune system and in the prevention of cancer. Apoptotic cells display characteristic changes in morphology that precede the eventual fragmentation of nuclear DNA resulting in cell death. Current evidence implicates DNase I as responsible for hydrolysis of DNA during apoptosis. In vivo, it is likely that cytoplasmic actin binds and inhibits the enzymatic activity and nuclear translocation of DNase I and that disruption of the actin-DNase I complex results in activation of DNase I. In this report we demonstrate that the N-terminal fragment of gelsolin (N-gelsolin) disrupts the actin-DNase I interaction. This provides a molecular mechanism for the role of the N-gelsolin in regulating DNase I activity. We also show that cofilin stabilises the actin-DNase I complex by forming a ternary complex that prevents N-gelsolin from releasing DNase I from actin. We suggest that both cofilin and gelsolin are essential in modulating the release of DNase I from actin.     

Subunit composition and Ca(2+)-ATPase activity of the vacuolar ATPase from barley roots.

The vacuolar ATPase was purified from a tonoplast-enriched membrane fraction from barley (Hordeum vulgare cv CM72) roots. The membranes were solubilized with Triton X-100 and the membrane proteins were separated by chromatography on Sephacryl S-400 followed by fast protein liquid chromatography on a Mono-Q column. The purified vacuolar ATPase was inhibited up to 90% by KNO3 or 80% by dicyclohexylcarbodiimide (DCCI). The ATPase was resolved into polypeptides of 115, 68, 53, 45, 42, 34, 32, 17, 13, and 12 kDa. An additional purification step of centrifugation on a glycerol gradient did not result in loss of any polypeptide bands or increased specific activity of the ATPase. Antibodies against the purified holoenzyme inhibited proton transport by the native ATPase. Two peaks of solubilized Ca(2+)-ATPase were obtained from the Sephacryl S-400 column. A peak of Ca(2+)-ATPase copurified with the vacuolar ATPase during all of the purification steps and was inhibited by NO3- and DCCI. It is proposed that this Ca(2+)-ATPase is a partial reaction of the plant vacuolar ATPase. The second Ca(2+)-ATPase was greatly retarded on the Sephacryl S-400 column and eluted after the main protein peak. It was not inhibited significantly by NO3- or DCCI. The second Ca(2+)-ATPase is a major component of ATP hydrolysis by the native membranes.     

Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments

We describe herein the purification of a protein from skeletal muscle that binds to ("caps") the morphologically defined barbed end of actin filaments. This actin-capping protein appeared to be a heterodimer with chemically and immunologically distinct subunits of Mr = 36,000 (alpha) and 32,000 (beta), Rs = 37 A, s20,w = 4.0 S, and a calculated native molecular weight of approximately 61,000. The protein was obtained in milligram quantities at greater than 95% purity from acetone powder of chicken skeletal muscle by extraction in 0.6 M KI, precipitation with ammonium sulfate, sequential chromatographic steps on DEAE-cellulose, hydroxylapatite, and Sephacryl S-200, followed by preparative rate zonal sucrose density gradient centrifugation. In immunoblots of myofibrillar proteins, affinity-purified antibodies selectively recognized protein bands of the same molecular weight as the subunits of the capping protein to which they were made, indicating that the isolated capping protein is a native myofibrillar protein, and not a proteolytic digestion product of a larger muscle protein. A specific interaction of the capping protein with the barbed end of actin filaments was indicated by its ability to inhibit actin filament assembly nucleated by spectrin-band 4.1-actin complex in 0.4 mM Mg2+, accelerate actin filament formation and increase the critical concentration of actin in 2-5 mM Mg2+, 75-100 mM KCl, and inhibit the addition of actin monomers to the barbed end of heavy meromyosin-decorated actin filaments as determined by electron microscopy. All of these effects occurred at nanomolar concentrations of capping protein and micromolar concentrations of actin, suggesting a high affinity interaction.     

Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I

A new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, was purified from the bovine synaptosomal membrane. The binding of calmodulin to this protein was Ca2+-dependent, and was inhibited by trifluoperazine, the association constant being calculated to be 2.2 X 10(6) M-1. Maximally, 1 mol of calmodulin bound to 1 mol of the purified protein. This protein was phosphorylated by both kinase II (Ca2+- and calmodulin-dependent kinase) and cyclic AMP-dependent kinase. In addition, antibody against this protein was demonstrated to have an immunological crossreactivity with synapsin I in the synaptosomal membrane.     

Cytoplasmic gels from macrophages. Evidence for the involvement of four proteins in the calcium-dependent solation of actin networks

A method has been devised to study the influence of Ca2+ on the in vitro formation of actin gel networks. Under appropriate conditions low-Ca2+ cytosolic extracts (less than 1 nM) from macrophages rapidly formed a macromolecular complex composed of actin, filamin, alpha-actinin and two new proteins of 70 kDa and 55 kDa. [Pacaud, M. (1986) Eur. J. Biochem. 156, 521-530]. Increasing concentrations of free Ca2+ to 1-2 microM resulted in complete inhibition of the association of 70-kDa protein, a protein which associates actin filaments into parallel arrays. Concentrations of Ca2+ greater than 3 microM caused incorporation of two additional proteins, gelsolin and a 18-kDa polypeptide, with no change in either the actin or alpha-actinin content of the cytoskeletal structures. Use of a polyacrylamide gel overlay technique with 125I-calmodulin revealed that a high-Mr calmodulin-binding protein analogous to spectrin was also associated with these structures when micromolar Ca2+ was present. Similar assays with 45CaCl2 indicated that the 70-kDa protein binds Ca2+ with high affinity. It is thus suggested that Ca2+ might regulate the dynamic assembly of microfilaments through several target proteins, gelsolin, the 70-kDa protein and calmodulin.     

Plant villin, lily P-135-ABP, possesses G-actin binding activity and accelerates the polymerization and depolymerization of actin in a Ca2+-sensitive manner

From germinating pollen of lily, two types of villins, P-115-ABP and P-135-ABP, have been identified biochemically. Ca(2+)-CaM-dependent actin-filament binding and bundling activities have been demonstrated for both villins previously. Here, we examined the effects of lily villins on the polymerization and depolymerization of actin. P-115-ABP and P-135-ABP present in a crude protein extract prepared from germinating pollen bound to a DNase I affinity column in a Ca(2+)-dependent manner. Purified P-135-ABP reduced the lag period that precedes actin filament polymerization from monomers in the presence of either Ca(2+) or Ca(2+)-CaM. These results indicated that P-135-ABP can form a complex with G-actin in the presence of Ca(2+) and this complex acts as a nucleus for polymerization of actin filaments. However, the nucleation activity of P-135-ABP is probably not relevant in vivo because the assembly of G-actin saturated with profilin, a situation that mimics conditions found in pollen, was not accelerated in the presence of P-135-ABP. P-135-ABP also enhanced the depolymerization of actin filaments during dilution-mediated disassembly. Growth from filament barbed ends in the presence of Ca(2+)-CaM was also prevented, consistent with filament capping activity. These results suggested that lily villin is involved not only in the arrangement of actin filaments into bundles in the basal and shank region of the pollen tube, but also in regulating and modulating actin dynamics through its capping and depolymerization (or fragmentation) activities in the apical region of the pollen tube, where there is a relatively high concentration of Ca(2+).     

A novel 40,000 Da Ca2+-dependent actin modulator from bovine brain

A monomeric protein of Mr 40,000 that modulates the polymer state of actin has been isolated from bovine brain. When added either to preformed actin filaments or to monomeric actin, prior to polymerization, the modulator reduces the low-shear viscosity of F-actin provided that Ca2+ is present. The 40 kDa protein also inhibits the rate of actin polymerization. The inhibition is fully suppressed by removal of Ca2+ and restored by subsequent readdition of Ca2+, suggesting that the Ca2+-controlled interaction of actin with the 40 kDa modulator is freely reversible.     

Isolation and physico-chemical properties of a protein, included in an endotoxin from Yersinia pseudotuberculosis

A major protein of the endotoxin from Yersinia pseudotuberculosis was isolated from the complex lipid A--protein by treatment with SDS and triton X-100 followed by gel-chromatography on Sephacryl S-300. Protein has apparent molecular mass 40 kDa and alanine as N-terminal amino acid residue. CD and IR spectroscopy conformational changes of the protein molecule in the process of its isolation. The thermal and pH stabilities of the protein were investigated by the methods of intrinsic fluorescence and differential scanning microcalorimetry. The isolated protein revealed two thermal transitions (at 30-35 and 50-55 degrees C), which depend on Ca2+ concentration.     

Ca2+ control of actin gelation. Interaction of gelsolin with actin filaments and regulation of actin gelation

We elucidated the mechanism by which gelsolin, a Ca2+-dependent regulatory protein from lung macrophages, controls the network structure of actin filaments. In the presence of micromolar Ca2+, gelsolin bound Ca2+. The Ca2+-gelsolin complex reduced the apparent viscosity and flow birefringence of F-actin and the lengths of actin filaments viewed in the electron microscope. However, concentrations of gelsolin causing these alterations did not effect proportionate changes in the turbidity of actin filament solutions or in the quantity of nonsedimentable actin as determined by a radioassay. From these findings, we conclude that gelsolin shortens actin filaments without net depolymerization. Such an effect on the distribution of actin filament lengths led to the prediction that low concentrations of gelsolin would increase the critical concentration of actin-binding protein required for incipient gelation of actin filaments in the presence of Ca2+, providing an efficient mechanism for controlling actin network structure. We verified the prediction experimentally, and we estimated that the Ca2+-gelsolin complex effectively breaks the bond between actin monomers in filaments with a stoichiometry of 1:1. The effect of Ca2+-gelsolin complex on actin solation was rapid, independent of temperature between 0 degrees and 37 degrees C, and reversed by reducing the free Ca2+ concentration.     

Ca2+-dependent binding of severin to actin: a one-to-one complex is formed

Severin is a protein from Dictyostelium that severs actin filaments in a Ca2+-dependent manner and remains bound to the filament fragments (Brown, S. S., K. Yamamoto, and J. A. Spudich , 1982, J. Cell Biol., 93:205-210; Yamamoto, K., J. D. Pardee , J. Reidler , L. Stryer , and J. A. Spudich , 1982, J. Cell Biol. 95:711-719). Further characterization of the interaction of severin with actin suggests that it remains bound to the preferred assembly end of the fragmented actin filaments. Addition of severin in molar excess to actin causes total disassembly of the filaments and the formation of a high-affinity complex containing one severin and one actin. This severin -actin complex does not sever actin filaments. The binding of severin to actin, measured directly by fluorescence energy transfer, requires micromolar Ca2+, as does the severing and depolymerizing activity reported previously. Once bound to actin in the presence of greater than 1 microM Ca2+, severin is not released from the actin when the Ca2+ is lowered to less than 0.1 microM by addition of EGTA. Tropomyosin, DNase I, phalloidin, and cytochalasin B have no effect on the ability of severin to bind to or sever actin filaments. Subfragment 1 of myosin, however, significantly inhibits severin activity. Severin binds not only to actin filaments, but also directly to G-actin, as well as to other conformational species of actin.     

Isolation of Ca2+ sensitive proteins linked to the membrane fraction of the brain cortex and the spinal cord in pigs

Four Ca2+-sensitive proteins of respective subunit molecular weights 67 kDa, 37 kDa, 36 kDa and 32 kDa were purified from pig brain and spinal cord. Associated to the particulate fraction at millimolar concentrations of free Ca2+, they were solubilized using an EGTA-containing buffer and purified by a selective Ca2+-dependent precipitation. The 36 kDa protein is present in the tissues in a tetrameric form of (2 X 36 kDa + 2 X 13 kDa) and in a monomeric form. These proteins with the 37 kDa protein share the functional properties of the two well-known Ca2+-binding proteins, named calpactin I and calpactin II; they were able to interact with F-actin, brain spectrin (fodrin) and phosphatidylserine-liposomes in a Ca2+-dependent manner. The 67 kDa protein depolymerizes the actin filament in presence of Ca2+, it also binds to tubulin and to the neurofilament subunit NF-70, but not to brain spectrin. The 32 kDa protein does not share any association with F-actin and brain spectrin.     

Chicken skeletal muscle has three Ca2+-dependent proteinases

Chicken breast muscle has three Ca2+-dependent proteinases, two requiring millimolar Ca2+ (m-calpain and high m-calpain) and one requiring micromolar Ca2+ (mu-calpain). High m-calpain co-purifies with mu-calpain through successive DEAE-cellulose (steep gradient), phenyl-Sepharose, octylamine agarose, and Sephacryl S-300 columns, but elutes after mu-calpain when using a shallow KCl gradient to elute a DEAE-cellulose column. The mu- and m-calpains have 80 and 28 kDa polypeptides and are analogous to the mu- and m-calpains that have been purified from bovine, porcine and rabbit skeletal muscle. High m-calpain, which seems to be a new Ca2+-dependent proteinase, is still heterogeneous after the DEAE-cellulose column eluted with a shallow KCl gradient. Additional purification through two successive HPLC-DEAE columns and one HPLC-SW-4000 gel permeation column produces a fraction having six major polypeptides and 6-8 minor polypeptides on SDS-PAGE. A 74-76 kDa polypeptide in this fraction reacts in Western blots with monospecific, polyclonal anti-calpain antibodies that react with both the 80 kDa and the 28 kDa polypeptides of mu- or m-calpain. High m-calpain also is related to mu- and m-calpain in that it causes the same limited digestion of skeletal muscle myofibrils, has a similar pH optimum near pH 7.9-8.4, requires Ca2+ for activity, and reacts with the calpain inhibitor, calpastatin, and a variety of serine and cysteine proteinase inhibitors in a manner identical to mu- and m-calpain. High m-calpain differs from mu- and m-calpain in its elution off DEAE-cellulose columns and its requirement of 3800 microM Ca2+ for one-half maximal activity compared with 5.35 microM Ca2+ for mu-calpain and 420 microM Ca2+ for m-calpain. The physiological significance of high m-calpain in unclear. The presence of mu-calpain in chicken breast muscle suggests that all skeletal muscles contain both mu- and m-calpain, although the relative proportions of these two proteinases may vary in different species.     

Mechanism of interaction of Dictyostelium severin with actin filaments

Severin, a 40,000-dalton protein from Dictyostelium that disassembles actin filaments in a Ca2+ -dependent manner, was purified 500-fold to greater than 99% homogeneity by modifications of the procedure reported by Brown, Yamamoto, and Spudich (1982. J. Cell Biol. 93:205-210). Severin has a Stokes radius of 29 A and consists of a single polypeptide chain. It contains a single methionyl and five cysteinyl residues. We studied the action of severin on actin filaments by electron microscopy, viscometry, sedimentation, nanosecond emission anisotropy, and fluorescence energy transfer spectroscopy. Nanosecond emission anisotropy of fluoresence-labeled severin shows that this protein changes its conformation on binding Ca2+. Actin filaments are rapidly fragmented on addition of severin and Ca2+, but severin does not interact with actin filaments in the absence of Ca2+. Fluorescence energy transfer measurements indicate that fragmentation of actin filaments by severin leads to a partial depolymerization (t1/2 approximately equal to 30 s). Depolymerization is followed by exchange of a limited number of subunits in the filament fragments with the disassembled actin pool (t1/2 approximately equal to 5 min). Disassembly and exchange are probably restricted to the ends of the filament fragments since only a few subunits in each fragment participate in the disassembly or exchange process. Steady state hydrolysis of ATP by actin in the presence of Ca2+-severin is maximal at an actin: severin molar ratio of approximately 10:1, which further supports the inference that subunit exchange is limited to the ends of actin filaments. The observation of sequential depolymerization and subunit exchange following the fragmentation of actin by severin suggests that severin may regulate site-specific disassembly and turnover of actin filament arrays in vivo.     

Calcium-dependent interaction of actin filaments with actin binding protein in the presence of calmodulin and caldesmon

Caldesmon, calmodulin-, and actin-binding protein of chicken gizzard did not affect the process of polymerization of actin induced by 0.1 M KCl. Caldesmon binds to F-actin, thus inhibiting the gelation action of actin binding protein (ABP; filamin). Low shear viscosity and flow birefringence measurements revealed that in a system of calmodulin, caldesmon, ABP, and F-actin, gelation occurs in the presence of micromolar Ca2+ concentrations, but not in the absence of Ca2+. Electron microscopic observations showed the Ca2+-dependent formation of actin bundles in this system. These results were interpreted by the flip-flop mechanism: in the presence of Ca2+, a calmodulin-caldesmon complex is released from actin filaments on which ABP exerts its gelating action. On the other hand, in the absence of Ca2+, caldesmon remains bound to actin filaments, thus preventing the action of ABP.     

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.     

Mersalyl, a sulfhydryl reagent, alters the solubility of myosin and cytoskeletal proteins of human platelets

We have examined the effect of a mercurial sulfhydryl reagent, mersalyl, on the protein composition of cytoskeletons by SDS-polyacrylamide gel electrophoresis after treatment of human platelets with Triton X-100 (Triton) containing mersalyl and Ca2+, and have found that mersalyl alters the protein composition of cytoskeletons in a Ca2+-dependent manner. At 1 X 10(-7) M Ca2+, 0.2 mM mersalyl, which represents approximately the equivalent amount of sulfhydryl of platelet suspensions that we used, specifically made myosin insoluble. The amount of myosin in Triton-mersalyl residues was increased by increasing the Ca2+ concentration of Triton lysis buffer. Actin-binding protein, 235 kDa polypeptide and alpha-actinin-like protein were decreased in Triton residues by mersalyl at Ca2+ concentrations less than 1 X 10(-7) M, while these polypeptides in Triton residues were increased by mersalyl in the presence of more than 2 X 10(-7) M Ca2+. Electron microscopic study revealed the presence of thick filaments with an appearance similar to that of the thick filaments of platelet myosin. Thus, the modification with mersalyl of sulfhydryls of platelet polypeptides along with changes in Ca2+ concentrations within a physiological range leads to changes in solubility of, and filament formation of, myosin, actin and other cytoskeletal proteins.