Isolation and characterization of a high molecular weight actin-binding protein from Physarum polycephalum plasmodia

A high molecular weight actin-binding protein was isolated from the Physarum polycephalum plasmodia. The protein ( HMWP ) shares many properties with other high molecular weight actin-binding proteins such as spectrin, actin-binding protein from macrophages, and filamin. It has a potent activity to cross-link F-actin into a gel-like structure. Its cross-linking activity does not depend on calcium concentrations. Hydrodynamic studies have revealed that the protein is in the monomeric state of a polypeptide chain with molecular weight of approximately 230,000 in a high ionic strength solvent, while it self-associates into a dimer under physiological ionic conditions. Electron microscopic examinations of HMWP have shown that the monomer particle observed in a high ionic strength solvent is rod shaped with the two-stranded morphology very similar to that of spectrin. On the other hand, under physiological ionic conditions, the HMWP dimer shows the dumb-bell shape with two globular domains connected with a thin flexible strand.     

Toxofilin, a novel actin-binding protein from Toxoplasma gondii, sequesters actin monomers and caps actin filaments

Toxoplasma gondii relies on its actin cytoskeleton to glide and enter its host cell. However, T. gondii tachyzoites are known to display a strikingly low amount of actin filaments, which suggests that sequestration of actin monomers could play a key role in parasite actin dynamics. We isolated a 27-kDa tachyzoite protein on the basis of its ability to bind muscle G-actin and demonstrated that it interacts with parasite G-actin. Cloning and sequence analysis of the gene coding for this protein, which we named Toxofilin, showed that it is a novel actin-binding protein. In in vitro assays, Toxofilin not only bound to G-actin and inhibited actin polymerization as an actin-sequestering protein but also slowed down F-actin disassembly through a filament end capping activity. In addition, when green fluorescent protein-tagged Toxofilin was overexpressed in mammalian nonmuscle cells, the dynamics of actin stress fibers was drastically impaired, whereas green fluorescent protein-Toxofilin copurified with G-actin. Finally, in motile parasites, during gliding or host cell entry, Toxofilin was localized in the entire cytoplasm, including the rear end of the parasite, whereas in intracellular tachyzoites, especially before they exit from the parasitophorous vacuole of their host cell, Toxofilin was found to be restricted to the apical end.     

Fragmin60 encodes an actin-binding protein with a C2 domain and controls actin Thr-203 phosphorylation in Physarum plasmodia and sclerotia

We report the isolation of a cDNA clone encoding a 60-kDa protein termed fragmin60 that cross-reacts with fragmin antibodies. Unlike other gelsolin-related proteins, fragmin60 contains a unique N-terminal domain that shows similarity with C2 domains of aczonin, protein kinase C, and synaptotagmins. The fragmin60 C2 domain binds three calcium ions, one with nanomolar affinity and two with micromolar affinity. Actin binding by fragmin60 requires higher calcium concentrations than does binding of actin by a fragmin60 mutant lacking the C2 domain, suggesting that the C2 domain secures the actin binding moiety in a conformation preventing actin binding at low calcium concentrations. The fragmin60 C2 domain does not bind phospholipids but interacts with the endogenous homologue of Saccharomyces cerevisiae S-phase kinase-associated protein (Skp1), as shown by pull-down assays and co-expression in mammalian cells. Recombinant fragmin60 promotes in vitro phosphorylation of actin Thr-203 by the actin-fragmin kinase. We further show that in vivo phosphorylation of actin in the fragmin60-actin complex occurs in sclerotia, a dormant stage of Physarum development, as well as in plasmodia. Our findings indicate that we have cloned a novel type of gelsolin-related actin-binding protein that is involved in controlling regulation of actin phosphorylation in vivo.     

Myosin heavy-chain kinase A from Dictyostelium possesses a novel actin-binding domain that cross-links actin filaments

Myosin heavy-chain kinase A (MHCK A) catalyses the disassembly of myosin II filaments in Dictyostelium cells via myosin II heavy-chain phosphorylation. MHCK A possesses a 'coiled-coil'-enriched domain that mediates the oligomerization, cellular localization and actin-binding activities of the kinase. F-actin (filamentous actin) binding by the coiled-coil domain leads to a 40-fold increase in MHCK A activity. In the present study we examined the actin-binding characteristics of the coiled-coil domain as a means of identifying mechanisms by which MHCK A-mediated disassembly of myosin II filaments can be regulated in the cell. Co-sedimentation assays revealed that the coiled-coil domain of MHCK A binds co-operatively to F-actin with an apparent K(D) of approx. 0.5 muM and a stoichiometry of approx. 5:1 [actin/C(1-498)]. Further analyses indicate that the coiled-coil domain binds along the length of the actin filament and possesses at least two actin-binding regions. Quite surprisingly, we found that the coiled-coil domain cross-links actin filaments into bundles, indicating that MHCK A can affect the cytoskeleton in two important ways: (1) by driving myosin II-filament disassembly via myosin II heavy-chain phosphorylation, and (2) by cross-linking/bundling actin filaments. This discovery, along with other supporting data, suggests a model in which MHCK A-mediated bundling of actin filaments plays a central role in the recruitment and activation of the kinase at specific sites in the cell. Ultimately this provides a means for achieving the robust and highly localized disruption of myosin II filaments that facilitates polarized changes in cell shape during processes such as chemotaxis, cytokinesis and multicellular development.     

Structure of macrophage actin-binding protein molecules in solution and interacting with actin filaments

We have examined the structure of actin-binding molecules in solution and interacting with actin filaments. At physiological ionic strength, actin-binding protein has a M_r value of 540 × 10³ as determined by direct and indirect hydrodynamic measurements. It is an asymmetrical dimer composed of 270 × 10³ dalton subunits. Viewed in the electron microscope after negative staining or low angle shadowing, actin-binding protein molecules assume a broad range of conformations varying from closed circular structures to fully extended strands 162 nm in contour length. All configurations are apparently derived from the same structure which consists of two monomer chains connected end-to-end. The radius of gyration determined from the electron microscopic images was 21.3 nm in agreement with the value of 17.6 nm calculated from hydrodynamic assays. The average axial ratio from hydrodynamic measurements was 17:1, whereas fully extended dimer molecules in the electron microscope would have an axial ratio of 54:1. All of these observations indicate that actin-binding protein dimers are extremely flexible. The flexibility parameter λ (Landau &; Lifshits, 1958) for actinbinding protein is 0.18 nm^?1.As determined by sedimentation, actin-binding protein binds to actin filaments with a K_a value of 2 × 10⁶m^?1 and a capacity of one dimer to 14 actin monomers in filaments. After incubation of high concentrations (molar ratio to actin ≥ 1:10) of actin-binding protein with actin filaments, long filament bundles are visible in the electron microscope. Under these conditions, actin-binding protein molecules decorate the actin filaments in the bundles at regular 40 nm intervals or once every 15 monomers, approximately equivalent to the binding capacity measured by sedimentation. Low concentrations of actin-binding protein (molar ratio to actin ≥ 1:50) which promote the gelation of actin filaments in solution, did not detectably alter the isotropy of the actin filaments. Direct visualization of actinbinding protein molecules between actin filaments in the electron microscope showed that dimers are sufficient for crossbridging of actin filaments and that actinbinding protein dimers are bipolar, composed of monomers connected head-to-head and having actin-binding sites located on the free tails.We conclude that actin-binding protein is a dimer at physiological ionic strength. Each dimer has two actin filament binding sites and is therefore sufficient to gel actin filaments in solution. The length and flexibility of the actin-binding protein subunits render this molecule structurally suited for the crosslinking of large helical filaments into isotropic networks.     

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.     

Rabbit alveolar macrophages contain a Ca2+-sensitive, 41,000-dalton protein which reversibly blocks the "barbed" ends of actin filaments but does not sever them

A 41,000-dalton Ca2+-sensitive actin-modulating protein has been purified from rabbit alveolar macrophages using ion exchange and gel filtration chromatography. On sodium dodecyl-polyacrylamide gel electrophoresis, this macrophage protein migrates more rapidly than actin and fails to cross-react with polyclonal anti-actin antibody. It has a Stokes radius of 3.0 nm and an isoelectric point of 6.6. In the presence of micromolar Ca2+ this 41,000-Da protein: reduces the viscosity of polymerized actin, nucleates actin filament assembly, causes a nearly instantaneous increase in fluorescence intensity of subcritical concentrations of pyrenyl-actin (estimated KD of the pyrene actin-macrophage protein complex, 5 X 10(-8) M), increases the critical concentration of actin by 0.65 microM (molar ratios of protein/actin, 1/100-1/10), blocks actin monomer depolymerization from the "barbed" filament ends, and does not sever preformed actin filaments. The ability of this protein to block filament ends is rapidly and completely inhibited by lowering free calcium ion concentration below the micromolar range.     

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.     

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.     

Characterization of the myosin light chain kinase from smooth muscle as an actin-binding protein that assembles actin filaments in vitro

In addition to its kinase activity, myosin light chain kinase has an actin-binding activity, which results in bundling of actin filaments [Hayakawa et al., Biochem. Biophys. Res. Commun. 199, 786-791, 1994]. There are two actin-binding sites on the kinase: calcium- and calmodulin-sensitive and insensitive sites [Ye et al., J. Biol. Chem. 272, 32182-32189, 1997]. The calcium/calmodulin-sensitive, actin-binding site is located at Asp2-Pro41 and the insensitive site is at Ser138-Met213. The cyanogen bromide fragment, consisting of Asp2-Met213, is furnished with both sites and is the actin-binding core of myosin light chain kinase. Cross-linking between the two sites assembles actin filaments into bundles. Breaking of actin-binding at the calcium/calmodulin-sensitive site by calcium/calmodulin disassembles the bundles.     

The architecture of actin filaments and the ultrastructural location of actin-binding protein in the periphery of lung macrophages

A highly branched filament network is the principal structure in the periphery of detergent-extracted cytoskeletons of macrophages that have been spread on a surface and either freeze or critical point dried, and then rotary shadowed with platinum-carbon. This array of filaments completely fills lamellae extended from the cell and bifurcates to form 0.2-0.5 micron thick layers on the top and bottom of the cell body. Reaction of the macrophage cytoskeletons with anti-actin IgG and with anti-IgG bound to colloidal gold produces dense staining of these filaments, and incubation with myosin subfragment 1 uniformly decorates these filaments, identifying them as actin. 45% of the total cellular actin and approximately 70% of actin-binding protein remains in the detergent-insoluble cell residue. The soluble actin is not filamentous as determined by sedimentation analysis, the DNAase I inhibition assay, and electron microscopy, indicating that the cytoskeleton is not fragmented by detergent extraction. The spacing between the ramifications of the actin network is 94 +/- 47 nm and 118 +/- 72 nm in cytoskeletons prepared for electron microscopy by freeze drying and critical point drying, respectively. Free filament ends are rare, except for a few which project upward from the body of the network or which extend down to the substrate. Filaments of the network intersect predominantly at right angles to form either T-shaped and X-shaped overlaps having striking perpendicularity or else Y-shaped intersections composed of filaments intersecting at 120-130 degrees angles. The actin filament concentration in the lamellae is high, with an average value of 12.5 mg/ml. The concentration was much more uniform in freeze-dried preparations than in critical point-dried specimens, indicating that there is less collapse associated with the freezing technique. The orthogonal actin network of the macrophage cortical cytoplasm resembles actin gels made with actin-binding protein. Reaction of cell cytoskeletons and of an actin gel made with actin- binding protein with anti-actin-binding protein IgG and anti-IgG-coated gold beads resulted in the deposition of clusters of gold at points where filaments intersect and at the ends of filaments that may have been in contact with the membrane before its removal with detergent. In the actin gel made with actin-binding protein, 75% of actin-fiber intersections labeled, and the filament spacing between intersections is consistent with that predicted on theoretical grounds if each added actin-binding protein molecule cross-links two filaments to form an intersection in the gel.(ABSTRACT TRUNCATED AT 400 WORDS)     

The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts.

Vasoactive agents which elevate either cGMP or cAMP inhibit platelet activation by pathways sharing at least one component, the 46/50 kDa vasodilator-stimulated phosphoprotein (VASP). VASP is stoichiometrically phosphorylated by both cGMP-dependent and cAMP-dependent protein kinases in intact human platelets, and its phosphorylation correlates very well with platelet inhibition caused by cGMP- and cAMP-elevating agents. Here we report that in human platelets spread on glass, VASP is associated predominantly with the distal parts of radial microfilament bundles and with microfilaments outlining the periphery, whereas less VASP is associated with a central microfilamentous ring. VASP is also detectable in a variety of different cell types including fibroblasts and epithelial cells. In fibroblasts, VASP is concentrated at focal contact areas, along microfilament bundles (stress fibres) in a punctate pattern, in the periphery of protruding lamellae, and is phosphorylated by cGMP- and cAMP-dependent protein kinases in response to appropriate stimuli. Evidence for the direct binding of VASP to F-actin is also presented. The data demonstrate that VASP is a novel phosphoprotein associated with actin filaments and focal contact areas, i.e. transmembrane junctions between microfilaments and the extracellular matrix.     

A calcium- and pH-regulated protein from Dictyostelium discoideum that cross-links actin filaments

We have purified an actin binding protein from amebas of Dictyostelium discoideum which we call 95,000-dalton protein (95K). This protein is rod shaped, approximately 40 nm long in the electron microscope, contains two subunits measuring 95,000 daltons each, and cross-links actin filaments. Cross-linking activity was demonstrated by using falling-ball viscometry, Ostwald viscometry, and electron microscopy. Cross-linking activity is optimal at 0.1 microM Ca++ and pH 6.8, but is progressively inhibited at higher Ca++ and pH levels over a physiological range. Half-maximal inhibition occurs at 1.6 microM free Ca++ and pH 7.3, respectively. Sedimentation experiments demonstrate that elevated Ca++ and pH inhibit the binding of 95K to F-actin which explains the loss of cross-linking activity. Electron microscopy demonstrates that under optimal conditions for cross-linking, 95K protein bundles actin filaments and that this bundling is inhibited by microM Ca++. Severing of actin filaments by 95K was not observed in any of the various assays under any of the solution conditions used. Hence, 95K protein is a rod-shaped, dimeric, Ca++- and pH-regulated actin binding protein that cross-links but does not sever actin filaments.     

Isolation of a novel 190 kDa protein from tobacco BY-2 cells: possible involvement in the interaction between actin filaments and microtubules

Interaction between actin filaments (AFs) and microtubules (MTs) has been reported in various plant cells, and the presence of a factor(s) connecting these two cytoskeletal networks has been suggested, but its molecular entity has not been elucidated yet. We obtained a fraction containing MT-binding polypeptides, which induced bundling of AFs and of MTs. A 190 kDa polypeptide which associated with AFs was selectively isolated from the fraction. This polypeptide was thought to have an ability to bind to both AFs and MTs. We raised a monoclonal antibody against the 190 kDa polypeptide. Immunostaining demonstrated the association of the 190 kDa polypeptide with AF bundles and with MT bundles formed in vitro. Immunocytochemical studies throughout the cell cycle revealed that the 190 kDa polypeptide was localized in the nucleus before nuclear envelope breakdown, and in the spindle and the phragmoplast during cell division. After the re-formation of the nuclear envelope, the 190 kDa polypeptide was sequestered to the daughter nuclei. Using the antibody, we succeeded in cloning a cDNA encoding the 190 kDa polypeptide.     

Characterization of a 36,000-dalton protein from the surface of Madin- Darby canine kidney cells involved in cell attachment and spreading

We have identified and immunochemically characterized a 36,000-dalton membrane glycoprotein from Madin-Darby canine kidney cells. This protein is surface-labeled by lactoperoxidase-mediated iodination and metabolically labeled by [35S]methionine. It binds to Concanavalin A and incorporates 2-D-3H-mannose residues, thus indicating it is a glycoprotein. Rabbit polyclonal antibodies against this protein evenly decorate the external surface of trypsinized, unpolarized cells. The external apical surface of confluent monolayers, grown under culture conditions in which the tight junctions are closed and the cells have acquired polarity, is also evenly stained. The basolateral aspects of the external surface are stained only when the tight junctions are opened by removal of Ca++ or when the antibody has access to the monolayer from the basal side, which indicates an even distribution of this antigen on the surface of polarized cells. The antibody has no inhibitory effect on the opening and resealing of tight junctions in dense cultures, but does inhibit the attachment and spreading of cells on a substrate, which then blocks the establishment of a confluent functional monolayer.     

Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein "insertin". Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 x 10(6) M-1 s-1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.     

Formation of 100 A filaments from purified glial fibrillary acidic protein in vitro.

Glial fibrillary acidic protein, which is specific to astroglia in the central nervous system, polymerizes in vitro into filaments similar to native ~ 100 Å filaments. Following purification from aqueous extracts of bovine brain by immunoaffinity chromatography, GFA ² protein is highly soluble in very low ionic strength solutions. Sedimentation equilibrium analysis of protein solutions in prefilament solvent conditions (2 mm-Tris · HCl, pH 7.8, 20 °C, containing 0.5 mm-dithiothreitol) indicates a paucidisperse mixture of species in solution with a typical range of apparent weight-average molecular weights from about 186,000 to 227,000. Between pH 6.0 and 8.0 the solubility is a function of pH and ionic strength as well as temperature, and precipitation is favored by lowering the pH or temperature and by raising the ionic strength. GFA protein associates in the form of filaments over a narrow range of pH and ionic strength; optimal conditions for polymerization of a 0.1 mg/ml protein solution are 100 mm-imidazole-HCl buffer (pH 6.8), at a temperature of 37 °C, and there is no requirement for co-factors. Filaments appear primarily as tangles of smooth curvilinear structures approximately 100 Å in diameter and of indefinite length, although some lateral association of filaments into thick bundles is also observed. While the formation of filaments is not affected by the presence or absence of reducing agent, under oxidizing conditions disulfide linkages form between protein subunits. Disassembly is achieved by dialysis against 2 mm-Tris · HCl buffer (pH 8.5), but this process is significantly enhanced by the addition of 0.5 mM-dithiothreitol during assembly and disassembly.These experiments clarify the role of GFA protein as the subunit of astroglialspecific intermediate filaments. In addition, they suggest that the 100 Å filament, as other components of the cytoskeleton, may assemble and disassemble in the glial cytoplasm.     

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.     

A 40,000-dalton protein from Dictyostelium discoideum affects assembly properties of actin in a Ca2+-dependent manner

A 40,000-dalton protein that affects the assembly properties of actin in a Ca2+-dependent manner has been purified from Dictyostelium discoideum. Gel filtration chromatography indicates that the native form of this protein is a monomer. A major effect of this protein is to reduce the sedimentability of F-actin in a stoichiometric fashion. Nearly complete loss of sedimentability is observed at ratios of the 40,000-dalton protein to actin of greater than 1:10. At low stoichiometries, this protein can accelerate the rate of actin assembly under certain experimental conditions. These effects of the 40,000- dalton protein on the actin assembly properties described above require calcium ion. The 40,000-dalton protein does not exert its effects by proteolyzing actin. Furthermore, peptide maps demonstrate that this protein is not a proteolytic fragment of actin.     

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.