Chick brain actin and myosin. Isolation and characterization

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.     

Isolation and characterization of covalently cross-linked actin dimer

Covalently cross-linked actin dimer was isolated from rabbit skeletal muscle F-actin reacted with phenylenebismaleimide (Knight, P., and Offer, G. (1978) Biochem. J. 175, 1023-1032). The UV spectrum of the purified cross-linked actin dimer, in a nonpolymerizing buffer, was very similar to that of native F-actin and not to the spectrum of G-actin. Cross-linked actin dimer polymerized to filaments that were indistinguishable in the electron microscope from F-actin made from native G-actin and that were similar to native F-actin in their ability to activate the Mg2+-ATPase of myosin subfragment-1. The critical concentrations of polymerization of cross-linked actin dimer in 0.5 mM and 2.0 mM MgCl2, 2 to 4 microM, and 1 to 2 microM, respectively, were similar to the values for native G-actin. Cross-linked actin dimer contained 2 mol of bound nucleotide/mol of dimer. One bound nucleotide exchanged with ATP in solution with a t 1/2 of 55 min and with ADP with a t 1/2 of 5 h. The second bound nucleotide exchanged much more slowly. The more rapidly exchangeable site contained 10 to 15% bound ADP.Pi and 85 to 90% bound ATP while the second site contained much less, if any, bound ADP.Pi. Cross-linked actin dimer had an ATPase activity in 0.5 mM MgCl2 that was 7 times greater than the ATPase activity of native G-actin and that was also stimulated by cytochalasin D. These data are discussed in relation to the possible role of ATP in actin polymerization and function with the speculation that the cross-linked actin dimer may serve simultaneously as a useful model for each of the two different ends of native F-actin.     

Partially polymerized erythrocyte actin obtained by affinity chromatography on DNAse sepharose. Comparison with rabbit skeletal muscle actin and role of calcium

A new procedure, consisting of affinity chromatography on DNAse sepharose, is worked out for the purification of human erythrocyte actin from an extract of acetone powder. Comparison of skeletal muscle and erythrocyte actin purified either by reversible polymerization or affinity chromatography on DNAse Sepharose led us to infer that the erythrocyte actin isolated by affinity chromatography was pure, devoid of spectrin, and was obtained in part under polymerized (di and tetrameric) forms. This partial polymerization is related to a loss of calcium bound to actin.     

Beta-actinin is not distinguishable from an actin barbed-end capping protein in chicken breast muscle

beta-Actinin is an actin-pointed end capping protein in skeletal muscle. Casella et al. have reported that a protein isolated from muscle acetone powder by procedures similar to those used for beta-actinin purification caps the barbed end of an actin filament (J. Biol. Chem. 261, 10915-10921 (1986)). We have confirmed the above results. However, it turned out that the two proteins were identical as to subunit sizes, peptide maps, and cross-reactivities with anti-beta-actinin IgG. The binding of the two proteins to opposite ends of an actin filament remains unexplained.     

Probing actin polymerization by intermolecular cross-linking

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.     

Comparison of the properties of two kinds of preparations of human blood platelet actin with sarcomeric actin

A new procedure of purification of actin from human blood platelets was used. This method starting from acetone powder of whole platelets gives a much higher yield than the one previously described (actin I) (Landon et al. (1977) Eur. J. Biochem., 81, 571-577). This actin II preparation has the same reduced viscosity as skeletal muscle actin, while the reduced viscosity of actin I preparation is about 1/10 of this value. Moreover actin I has the form of very short filaments as shown by electron microscopy. After an extra step of purification actin I, when polymerized, acquired a high reduced viscosity. We confirmed that platelet and sarcomeric actins are similar in their polymerization properties and their ability to activate muscular myosin. A circular dichroism study showed that the overall conformation of both actins are similar, but the environment of their aromatic chromophores is different.     

Actin in embryonic organ epithelia

The presence of actin in morphogenetically active embryonic epithelia was assessed by SDS-polyacrylamide gel electrophoresis of pellets and low ionic strength extracts of acetone powders of isolated mouse embryo salivary and lung epithelia, and chick embryo epidermis. A polypeptide that co-electrophoresed with skeletal muscle actin was resolved in each of these systems. Approx. 80–95% of this protein was extracted from acetone powders by low ionic strength solutions, demonstrating solubility properties like those of muscle actin. Similar results were obtained with salivary, bronchial, and pancreatic mesenchyme. Cytoplasmic actin represented approx. 7 % of the protein in salivary epithelium and 13% of the protein in salivary mesenchyme. Electron microscopy demonstrated that incubation of glycerinated salivaries in low ionic strength solutions preferentially removed microfilaments from the epithelia, thus linking these heavy meromyosin-binding structures with the actin extracted from acetone powders and resolved on SDS-gels.     

Purification and characterization of actobindin, a new actin monomer-binding protein from Acanthamoeba castellanii

Actobindin is a new actin-binding protein isolated from Acanthamoeba castellanii. It is composed of two possibly identical polypeptide chains of approximately 13,000 daltons, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and with isoelectric points of 5.9. In the native state, actobindin appears to be a dimer of about 25,000 daltons by sedimentation equilibrium analysis. It contains no tryptophan and probably no tyrosine. Actobindin reduces the concentration of F-actin at steady state and inhibits the rate of filament elongation to extents consistent with the formation of a 1:1 actobindin-G-actin complex in a reaction with a KD of about 5 microM. The available data do not eliminate the possibility of other stoichiometries for the complex, but they are not consistent with any significant interaction between actobindin and F-actin. Despite the similarities between the effects of actobindin and Acanthamoeba profilin on the polymerization of Acanthamoeba actin, the two proteins are quite distinct with different native and subunit molecular weights, different isoelectric points, and different amino acid compositions. Also, unlike profilin, actobindin binds as well to rabbit skeletal muscle G-actin and to pyrenyl-labeled G-actin as it does to unmodified Acanthamoeba G-actin.     

In vitro filament formation of a new fiber-forming protein from Tetrahymena pyriformis

Using a 38,000-dalton protein (FFP-38) purified from Tetrahymena acetone powder, we have succeeded in the polymerization of this protein into 14-nm filaments. The polymerization was initiated by incubating the purified FFP-38 fraction in a buffer containing 5 mM Mes (2-(N-Morpholino)ethanesulfonic acid), 50 mM KCl, 1.2 mM CaCl2, 0.6 mM ATP, pH 6.6, and by shifting the incubation temperature from 0 degrees C to 37 degrees C. The 14-nm filament is considered to consist of 7-nm globular subunits regularly arranged into 2 start, helical strands with 4 subunits per turn. The subunit may correspond to 9S tetramer of FFP-38, a native form of FFP-38. Since the subunit arrangement and subunit protein component of this 14-nm filament obviously differ from those of actin filament, 10-nm intermediate filament and microtubule, the 14-nm filament appears to be a newly found intracellular filament. Concerning the FFP-38 polymerization, some polymorphism appeared: we found ring structures having the diameters of 0.3--3.7 micrometers and latticed sheet structure, besides typical straight filaments.     

THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg⁺⁺ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state.     

Bovine aorta actin. Development of an improved purification procedure and comparison of polymerization properties with actins from other types of muscle

Crude actin extracts from acetone-dried powder of the muscle layer of bovine aorta contain an actin-modulating protein which promotes nucleation of actin monomers and decreases the average length of actin filaments in a Ca2+-dependent manner. This observation has allowed the development of an improved purification procedure for aorta actin which increases the yield 2- to 3-times. The actin obtained with this procedure consists of 77% alpha- and 23% gamma-isoelectric species. Pure aorta actin is indistinguishable from actins from skeletal, cardiac and chicken-gizzard smooth muscle in its polymerization rate, critical concentration, and reduced viscosity when polymerized with KCl at 25 degrees C. It differs from sarcomeric actins, but not from chicken-gizzard smooth muscle actin, in the temperature dependence of polymerization equilibria in KCl. This difference correlates with the amino acid replacements Val-17----Cys-17 and Thr-89----Ser-89, supporting a conclusion drawn from other studies that the N-terminal portion of actin polypeptide chain contains sites important for polymerization.     

Polymerization of G-actin by myosin subfragment 1

The polymerization of actin from rabbit skeletal muscle by myosin subfragment 1 (S-1) from the same source was studied in the depolymerizing G-actin buffer. The polymerization reactions were monitored in light-scattering experiments over a wide range of actin/S-1 molar rations. In contrast to the well resolved nucleation-elongation steps of actin assembly by KC1 and Mg2+, the association of actin in the presence of S-1 did not reveal any lag in the polymerization reaction. Light scattering titrations of actin with S-1 and vice versa showed saturation of the polymerization reaction at stoichiometric 1:1 ratios of actin to S-1. Ultracentrifugation experiments confirmed that only stoichiometric amounts of actin were incorporated into a 1:1 acto-S-1 polymer even at high actin/S-1 ratios. These polymers were indistinguishable from standard complexes of S-1 with F-actin as judged by electron microscopy, light scattering measurements, and fluorescence changes observed while using actin covalently labeled with N-(1-pyrenyl)iodoacetamide. F-actin obtained by polymerization of G-actin by S-1 could initiate rapid assembly of G-actin in the presence of 10 mM KC1 and 0.5 mM MgCl2 and showed normal activation of MgATPase hydrolysis by myosin.     

Correlation between polymerizability and conformation in scallop beta-like actin and rabbit skeletal muscle alpha-actin.

In order to investigate the structural basis for functional differences among actin isoforms, we have compared the polymerization properties and conformations of scallop adductor muscle beta-like actin and rabbit skeletal muscle alpha-actin. Polymerization of scallop Ca(2+)-actin was slower than that of skeletal muscle Ca(2+)-actin. Cleavage of the actin polypeptide chain between Gly-42 and Val-43 with Escherichia coli protease ECP 32 impaired the polymerization of scallop Mg(2+)-actin to a greater extent than skeletal muscle Mg(2+)-actin. When monomeric scallop and skeletal muscle Ca(2+)-actins were subjected to limited proteolysis with trypsin, subtilisin, or ECP 32, no differences in the conformation of actin subdomain 2 were detected. At the same time, local differences in the conformations of scallop and skeletal muscle actin subdomains 1 were revealed as intrinsic fluorescence differences. Replacement of tightly bound Ca(2+) with Mg(2+) resulted in more extensive proteolysis of segment 61-69 of scallop actin than in the case of skeletal muscle actin. Furthermore, segment 61-69 was more accessible to proteolysis with subtilisin in polymerized scallop Ca(2+)-actin than in polymerized skeletal muscle Ca(2+)-actin, indicating that, in the polymeric form, the nucleotide-containing cleft is in a more open conformation in beta-like scallop actin than in skeletal muscle alpha-actin. We suggest that this difference between scallop and skeletal muscle actins is due to a less efficient shift of scallop actin subdomain 2 to the position it has in the polymer. The possible consequences of amino acid substitutions in actin subdomain 1 in the allosteric regulation of the actin cleft, and hence in the different stabilities of polymers formed by different actins, are discussed.     

Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase

Catecholamines are known to influence the contractility of cardiac and skeletal muscles, presumably via cAMP-dependent phosphorylation of specific proteins. We have investigated the in vitro phosphorylation of myofibrillar proteins by the catalytic subunit of cAMP-dependent protein kinase of fast- and slow-twitch skeletal muscles and cardiac muscle with a view to gaining a better understanding of the biochemical basis of catecholamine effects on striated muscles. Incubation of canine red skeletal myofibrils with the isolated catalytic subunit of cAMP-dependent protein kinase and Mg-[gamma-32P]ATP led to the rapid incorporation of [32P]phosphate into five major protein substrates of subunit molecular weights (MWs) 143,000, 60,000, 42,000, 33,000, and 11,000. The 143,000 MW substrate was identified as C-protein; the 42,000 MW substrate is probably actin; the 33,000 MW substrate was shown not to be a subunit of tropomyosin and, like the 60,000 and 11,000 MW substrates, is an unidentified myofibrillar protein. Isolated canine red skeletal muscle C-protein as phosphorylated to the extent of approximately 0.5 mol Pi/mol C-protein. Rabbit white skeletal muscle and bovine cardiac muscle C-proteins were also phosphorylated by the catalytic subunit of cAMP-dependent protein kinase, both in myofibrils and in the isolated state. Cardiac C-protein was phosphorylated to the extent of 5-6 mol Pi/mol C-protein, whereas rabbit white skeletal muscle C-protein was phosphorylated at the level of approximately 0.5 mol Pi/mol C-protein. As demonstrated earlier by others, C-protein of skeletal and cardiac muscles inhibited the actin-activated myosin Mg2+-ATPase activity at low ionic strength in a system reconstituted from the purified skeletal muscle contractile proteins (actin and myosin).(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.     

On the interaction of bovine seminal RNase with actin in vitro

Ribonuclease from bovine seminal plasma (RNase BS) interacts with skeletal muscle actin in the following way: it binds to actin with an apparent binding constant of 9.2 X 10(4) M-1 in 0.1 M KCl, induces the polymerization of actin below the critical concentration in depolymerization buffer, accelerates the salt-induced polymerization of actin even at a molar ratio of RNase to actin lower than 1/100, and bundles F-actin filaments. In the bundles the molar ratio of RNase to actin is about 0.66. Actin inhibits the enzymatic activity of RNase BS. RNase A from bovine pancreas, which is structurally almost identical to the subunits of RNase BS as well as a monomeric form of RNase BS, do not cross-link actin filaments and have a much smaller effect on the polymerization of actin. We conclude that the dimeric structure of the RNase BS, which consists of two identical subunits cross-linked by interchain disulfide bridges, is probably responsible for the bundling activity and the accelerating effect on the polymerization of actin.     

Isolation and immunofluorescent localization of actin derived from rat skeletal muscle

Summary Rat skeletal muscle actin was extracted, purified and its homogeneity established according to the criteria of ultracentrifugation and electrophoresis. Immunofluorescence procedure using antisera prepared in rabbits against the purified rat skeletal muscle actin revealed localized staining reaction in the I band region of the skeletal muscle. Similar studies on rat embryo muscle cultures showed a diffuse cytoplasmic fluorescence in fibroblastlike cells and an intense fluorescence in the multi-nucleated myoblasts of the younger cultures. In the older cultures strong fluorescence was detectable in scattered parallel rows and in the presumptive I bands of mononucleated myoblasts an in the thread-like mitochondria of fibroblasts. The distribution of fluorescence in these cells is considered indicative of the association of actin with the contracile protein in general and with mitochondria which in cultured myoblasts assume enormous lengths and appear to be extremely motile.     

Polymerization-induced changes in the fluorescence of actin labeled with iodoacetamidotetramethylrhodamine

Rabbit skeletal muscle actin has been labeled with the fluorescent sulfhydryl reagent iodoacetamidotetramethylrhodamine (rhodamine). The label is probably located on Cys-373 because prior treatment of the actin with N-ethylmaleimide prevents incorporation of rhodamine. When the rhodamine-actin is polymerized with MgSO₄ or KCl, there is approximately a 1.5-fold increase in fluorescence. The change in fluorescence is correlated with incorporation of monomeric globular actin into polymer and can therefore be used as a quantitative measure of polymerization. Trace quantities of rhodamine-actin can be used to follow the kinetics of polymerization of unlabeled actin without disturbing the sample, and it is shown that the use of a capillary viscometer accelerates the rate of polymerization. Fluorescence photobleaching recovery experiments, which measure the diffusion of rhodamine-labeled actin, show that nondiffusible (apparent diffusion coefficient < 10^?10 cm²/s) filaments appear during the polymerization process but that immediately after shearing these filaments are readily diffusible. These results demonstrate the destructive nature of hydrodynamic shear stress on polymerized actin.     

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.     

Eu-actinin, a new structural protein of the Z-line of striated muscles

A new protein component of the Z-line of striated muscles was isolated from chicken breast muscle. This protein has been designated as eu-actinin because of its close similarity in polypeptide molecular weight to actin. Eu-actinin was extracted from myosin-removed myofibrils at low ionic strength at pH 6.5 and purified by column chromatography on Sepharose 4B and DEAE-cellulose. Although the polypeptide molecular weight of eu-actinin measured by SDS-polyacrylamide gel electrophoresis is similar to that of actin, other physico-chemical properties of eu-actinin definitely differ from those of actin. The isoelectric point of eu-actinin was more acidic than that of actin. The amino acid composition of eu-actinin was found to be different from that of actin or those of other muscle structural proteins. The results of analytical gel filtration on Sepharose 4B indicated that eu-actinin forms dimers through non-covalent bonding under aqueous conditions. Eu-actinin has a low axial asymmetry under low-salt conditions, as judged from its intrinsic viscosity ([eta] = 6.4 ml/g for the dimer state) and exhibits a tendency to undergo self-association with increasing ionic strength. Interactions of eu-actinin with other muscle proteins were examined by the affinity column technique. It was shown that eu-actinin binds to actin and alpha-actinin. Eu-actinin exhibited strong seeding ability for the polymerization of actin. Antibody to eu-actinin was raised in a goat and purified by affinity chromatography. The specific antibody against eu-actinin did not form precipitine lines with actin or alpha-actinin. Immunofluorescence studies revealed that eu-actinin is localized at the Z-line of myofibrils. The FITC-conjugated antibody to eu-actinin also stained the Z-lines of rabbit skeletal muscle and chicken cardiac muscle. Therefore, it was concluded that eu-actinin is a new, ubiquitous constituent of Z-lines of striated muscles.