A fragmin-like protein from plasmodium of Physarum polycephalum that severs F-actin and caps the barbed end of F-actin in a Ca2+-sensitive way

Many protein factors regulating actin polymerization can be extracted from plasmodia of Physarum polycephalum in the presence of a high EGTA concentration (30 mM). A protein factor with the molecular weight of 60,000 (60 kDa protein) was especially interesting because of its fragmin-like properties. We purified and characterized this 60 kDa protein in the present study. The purified 60 kDa protein enhanced the initial rate of G-actin polymerization, severed F-actin, and capped the barbed end of F-actin in a Ca2+-dependent way. The threshold concentration for Ca2+ was around 10(-6) M. The flow birefringence measurement showed that the length of F-actin decreased from 2.8 to 1.0 microns depending on the concentration of 60 kDa protein added to F-actin. These properties were identical to those of fragmin (Mr 42,000) isolated from plasmodia (Hasegawa et al. (1980) Biochemistry 19, 2677-2683). However, the molecular weight, the tryptic peptide map, and the cross-reactivities with polyclonal anti-fragmin antibodies were different from those of fragmin. We concluded from these results that 60 kDa protein is a new Ca2+-sensitive F-actin-severing protein. Considering its similarity to fragmin, we termed the 60 kDa protein fragmin 60.     

Actin-fragmin interactions as revealed by chemical cross-linking

A one to one complex of actin and fragmin (a capping protein from Physarum polycephalum plasmodia) was cross-linked with 1-ethyl-3-[3-(dimethylamino)propyl] carbodiimide. The cross-linking reaction generated two cross-linked products with slightly different molecular weights (88 000 and 90 000) as major species. They were cross-linked products of one actin and one fragmin. The cross-linking site of fragmin in the actin sequence was determined by peptide mappings [Sutoh, K. (1982) Biochemistry 21, 3654-3661] after partial chemical cleavages of cross-linked products with hydroxylamine. The results indicated that the N-terminal segment of actin spanning residues 1-12 participated in cross-linking with fragmin. The cross-linker used in this study covalently bridges lysine side chains and side chains of acidic residues when they are in direct contact. Therefore, it seems that acidic residues in the N-terminal segment of actin (Asp-1, Glu-2, Asp-3, Glu-4, and Asp-11), at least some of them, are in the binding site of fragmin. It has already been shown that the same acidic segment of actin is in the binding site of myosin or depactin (an actin-depolymerizing protein isolated from starfish oocytes). We suggest that the unusual amino acid sequence of the N-terminal segment of actin makes its N-terminal region a favorable anchoring site for various types of actin-binding proteins.     

A novel 53 kDa actin binding protein from porcine brain--further biochemical and immunological characterization

We have previously described some of the characteristics of an actin binding protein, 53 K protein, purified from porcine brains. The purification procedure was revised in order to investigate of this actin binding protein further. A Scatchard plot analysis showed that the association constant between actin and the 53 K protein has around the same value as those reported for the fascin-actin and for the filamin-actin interactions. The binding experiments also demonstrated the occurrence of competitive binding with other actin binding proteins such as filamin, alpha-actinin, caldesmon and tropomyosin for the actin filament. Antibody was produced against brain 53 K protein and further purified on an affinity column. Immunoblot analysis using the antibody showed that this protein is localized in both the soluble and membraneous fraction of the brain. Other tissues such as liver and lung also contain 53 K protein. The immunoblot analysis also revealed that the gelation product of rat brain extract described by Palmer et al. contains immunoreactive polypeptides having slightly lower molecular weights and more basic isoelectric points than porcine brain 53 K protein. Immunological localization of the 53 K protein within HeLa and BS-C-1 cells showed that this protein is distributed throughout the cell in small granules, and in some regions of the cell, these granules were aggregated into much larger granules.     

A cofilin-like protein is involved in the regulation of actin assembly in developing skeletal muscle

An actin-binding protein of 20 kDa (called 20K protein) was purified from the sarcoplasmic fraction of embryonic chicken skeletal muscle. The properties of this protein were very similar to cofilin, which was discovered in porcine brain (Nishida et al. (1984) Biochemistry, 23, 5307-5313): it bound to both G- and F-actin, inhibited actin polymerization in a pH-dependent manner, inhibited binding of tropomyosin to F-actin, and had almost the same molecular size and pI as cofilin. A specific monoclonal antibody to 20K protein (MAB-22) was prepared to examine the expression and location of 20K protein during skeletal muscle development. When the whole protein lysates of embryonic and post-hatched chicken skeletal muscles were examined by means of immunoblotting combined with SDS-PAGE, 20K protein was detected in skeletal muscle through the developmental stages. Location of 20K protein in the cells differed between the embryonic and adult tissues; immunofluorescence staining of the cryosections of embryonic muscle with MAB-22 visualized irregular dot-like structures, but adult muscle sections were stained faintly and uniformly. 20K protein was present as a complex with actin in embryonic muscle, as judged by the ability to bind to a DNase I affinity column, while the same protein was free from actin in the cytoplasm of adult muscle. From these results, it is suggested that 20K protein regulates actin assembly transiently in developing skeletal muscle.     

Comparison of a major heat-stable microtubule-associated protein in HeLa cells and 190-kDa microtubule-associated protein in bovine adrenal cortex

A heat-stable microtubule-associated protein (MAP) with a molecular weight of 190,000, termed 190-kDa MAP, has been purified from bovine adrenal cortex (Murofushi, H. et al. (1986) J. Cell Biol. 103, 1911-1919). Immunoblotting experiments using an antibody against this MAP revealed that several kinds of culture cells derived from human tissues contain proteins with an apparent molecular weight of 180,000 reacting with the antibody. Indirect immunofluorescence microscopic observation of HeLa cells showed that the immunoreactive protein co-exists with microtubules, indicating that the protein is one of the HeLa MAPs. A heat-stable MAP with a molecular weight of 180,000, termed here HeLa 180-kDa MAP, was purified by the taxol-dependent procedure (Vallee, R.B. (1982) J. Cell Biol. 92, 435-442) and successive co-polymerization with brain tubulin. This protein was the most abundant MAP in HeLa cells, suggesting that the MAP is identical to the major HeLa MAP previously reported by Bulinski and Borisy (Bulinski, J.C. & Borisy, G.G. (1980) J. Biol. Chem. 255, 11570-11576) and Weatherbee et al. [1980) Biochemistry 19, 4116-4123). It was shown that, like bovine adrenal 190-kDa MAP, yet distinct from brain MAP2 and tau, purified HeLa 180-kDa MAP does not interact with actin filaments. This common characteristic of the two MAPs along with the same heat-stability strongly suggests that they are members of the same group of MAPs. The fact that HeLa 180-kDa MAP reacts with an antibody against bovine adrenal 190-kDa MAP means that they share common epitopes, in other words, common local amino acid sequences. However, the limited proteolytic patterns of the two MAPs with S. aureus V8 protease and chymotrypsin were distinct from each other, suggesting the presence of large differences in the overall primary structures between bovine adrenal 190-kDa MAP and HeLa 180-kDa MAP.     

Further characterization of a brain high molecular weight actin-binding protein (BABP): interaction with brain actin and ultrastructural studies

Crude actomyosin fraction from porcine brain contained a large amount of high molecular weight actin-binding protein (BABP). The molar ratio of BABP to actin (BABP/actin) in the fraction was estimated to be 0.22. From this fraction, BABP and actin were solubilized with a molar ratio of 0.25, suggesting the existence of an interaction between BABP and brain actin. BABP was finally purified to 90% purity. The purified BABP was negatively stained and observed by electron microscopy; it appeared to be a slender, flexible, two-stranded molecule whose contour length was about 200 nm. The structure was very similar to those of fodrin and other high molecular weight actin-binding proteins such as filamin, spectrin, and ABP. Lattice cage-like structures composed of BABP molecules were occasionally observed at high BABP concentrations. The addition of BABP to actin filaments resulted in the appearance of many branching, filamentous bundles. The electron microscopic observations suggested that a single BABP molecule could crosslink actin filaments, that is, one BABP molecule has two actin binding sites.     

Activity of the plant peptide aglycin in mammalian systems

A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27-63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 microg.g(-1) body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology.     

Comparison of Amino-acid Sequences of Hypothalamic Peptide,Brain-specific Histone and Myelin Basic Protein

SEVERAL studies have described the basic proteins of porcine central nervous tissue. Shome and Saffran¹ have isolated and purified a large peptide (or small protein) of molecular weight about 14,000 from acetone-dried hog hypothalamic powder. Sixteen of the approximately twenty-six tryptic digest fragments of this protein have been sequenced. The protein has no pressor or ACTH-releasing activity. Tomasi-and Kornguth² purified and partially characterized a basic protein from pig brain and, on the basis of fluorescent antibody studies, they concluded that this protein is a brain-specific histone, found in neurone nuclei (or nucleoli) of several animals^3–5.     

Binding of dicyclohexylcarbodiimide to a native F1-ATPase-inhibitor protein complex isolated from bovine heart mitochondria

The effect and the binding of dicyclohexylcarbodiimide (DCCD) to a soluble native F1-ATPase-inhibitor protein complex (F1-IP) isolated from heart mitochondria was studied. About one mol DCCD bound per mol F1-IP complex; this inhibited its ATPase activity by more than 95%, ever under conditions that led to maximal hydrolysis. Bound DCCD localized to beta-subunits of the F1-IP complex. Cross-linking of the DCCD labeled complex with N-(ethoxy-carbonyl)-2-ethoxydihydroquinoline yielded a protein with a Mr 65,000-67,000 that contained IP as evidenced by its reaction with IP antibodies. No alpha-subunits were detected in this cross-linked product. The Mr 65,000-67,000 protein corresponds to beta-subunits cross-linked with IP (Klein et al, Biochemistry 1980; 19, 2919-2925). However, no DCCD was found in the cross-linked beta-subunit-IP product of labeled native F1-IP. Thus the beta-subunit in contact with IP is distinct from the other two beta-subunits of the enzyme.     

Isolation of a Ca2+-Dependent Actin-Fragmenting Protein from Brain, Spinal Cord, and Cultured Neurones

Extracts of ox spinal cord and chicken brain were fractionated by ion-exchange chromatography and assayed for their ability to reduce the viscosity of muscle F-actin solutions. Two distinct peaks of activity were obtained, one of which was further purified by affinity chromatography on a DNAase-actin Sepharose column. Following molecular exclusion chromatography, the actin component appeared as a complex of 1 molecule of a protein with molecular weight 90,000 and 2 molecules of actin (42,000). This tightly bound complex was resistant to most methods of protein separation, but was resolvable into its component proteins by sodium dodecyl sulphate acrylamide gel electrophoresis. The protein of molecular weight 90,000 could be eluted from such a gel in a fully active form. The activity of the protein from ox spinal cord was closely similar to that of gelsolin, an actin-fragmenting protein originally isolated from rabbit lung macrophages. Like gelsolin, the protein from ox spinal cord produced fragmentation of muscle F-actin filaments at Ca2+ concentrations greater than 10(-7) M, and had a nucleating effect on the polymerisation of muscle actin; the latter was measured most easily by the enhancement of fluorescence of muscle actin conjugated to N-(1-pyrenyl)iodoacetamide. Nucleation was more effective in the presence of Ca2+, but also occurred in its absence, and the same was true of complex formation between the 90,000 protein and muscle G-actin. On the basis of its actin-fragmenting activity, we estimate that the 90,000 molecular weight protein constitutes 0.2% of the protein initially extracted from ox spinal cord. A very similar protein, indistinguishable in its action on actin but containing variable amounts of a protein of molecular weight 85,000 as well as 90,000, was isolated from chicken brain. A similar protein was also detected in pure cultures of sympathetic neurones by enrichment on a DNAase-actin affinity column and by immune blotting and by immunofluorescence. We conclude that a protein similar, if not identical to macrophage gelsolin is present in neurones and that it probably plays a part in the actin-based movements of these cells.     

Purification and characterization of an alpha-actinin-like protein from porcine kidney

An alpha-actinin-like protein was partially purified from the Triton-insoluble cytoskeleton of porcine kidney by 0.6 M MgCl2 treatment, ammonium sulfate fractionation, DEAE-cellulose chromatography and hydroxyapatite chromatography. Apparent purity of the kidney protein was approximately 90% by quantitative densitometry of Coomassie-stained polyacrylamide gels. The kidney alpha-actinin-like protein is very similar to muscle alpha-actinins by the following criteria: (1) both kidney protein and muscle alpha-actinins bind to F-actin at a similar ratio; (2) both proteins demonstrate no difference in the actomyosin turbidity assay and the ATPase assay for alpha-actinin activity; (3) both native proteins contain a large core of identical molecular weight resistant to trypsin; (4) on two-dimensional gels, both kidney protein and muscle alpha-actinins have similar isoelectric points of 5.9-6.1. However, kidney alpha-actinin-like protein is not identical in every respect with muscle alpha-actinins. Electrophoretic mobility of the kidney protein is slightly greater than that of chicken gizzard alpha-actinin and is identical to that of a component of chicken skeletal muscle alpha-actinin. One-dimensional peptide mappings of the kidney protein and muscle alpha-actinins were significantly different from each other. The interaction between kidney alpha-actinin-like protein and F-actin is sensitive to Ca2+. Similar Ca2+-sensitivity was observed with other non-muscle cell alpha-actinins.     

Purification of a high molecular weight actin filament gelation protein from Acanthamoeba that shares antigenic determinants with vertebrate spectrins

I have purified a high molecular weight actin filament gelation protein (GP-260) from Acanthamoeba castellanii, and found by immunological cross-reactivity that it is related to vertebrate spectrins, but not to two other high molecular weight actin-binding proteins, filamin or the microtubule-associated protein, MAP-2. GP-260 was purified by chromatography on DEAE-cellulose, selective precipitation with actin and myosin-II, chromatography on hydroxylapatite in 0.6 M Kl, and selective precipitation at low ionic strength. The yield was 1-2 micrograms/g cells. GP-260 had the same electrophoretic mobility in SDS as the 260,000-mol-wt alpha-chain of spectrin from pig erythrocytes and brain. Electron micrographs of GP-260 shadowed on mica showed slender rod-shaped particles 80-110 nm long. GP-260 raised the low shear apparent viscosity of solutions of Acanthamoeba actin filaments and, at 100 micrograms/ml, formed a gel with a 8 microM actin. Purified antibodies to GP-260 reacted with both 260,000- and 240,000-mol-wt polypeptides in samples of whole ameba proteins separated by gel electrophoresis in SDS, but only the 260,000-mol-wt polypeptide was extracted from the cell with 0.34 M sucrose and purified in this study. These antibodies to GP-260 also reacted with purified spectrin from pig brain and erythrocytes, and antibodies to human erythrocyte spectrin bound to GP-260 and the 240,000-mol-wt polypeptide present in the whole ameba. The antibodies to GP-260 did not bind to chicken gizzard filamin or pig brain MAP-2, but they did react with high molecular weight polypeptides from man, a marsupial, a fish, a clam, a myxomycete, and two other amebas. Fluorescent antibody staining with purified antibodies to GP-260 showed that it is concentrated near the plasma membrane in the ameba.     

Interaction of myosin subfragment-1 with actin. II. Location of the actin binding site in a fragment of subfragment-1 heavy chain

The heavy chain of subfragment-1 prepared by chymotrypsin treatment had a molecular weight of about 96K. The heavy chain was split into 26 K, 50 K, and 21 K fragments by trypsin. When the trypsin-treated subfragment-1 was cross-linked with dimethyl suberimidate, cross-linked products of 26 K, 50 K, and 21 K fragments and of 50 K and 21 K fragments appeared, but there was little cross-linked product of 26 K and 50 K fragments or of 26 K and 21 K fragments. When the cross-linking experiments were carried out in the presence of actin, a new band appeared and the amount of cross-linked product of 26 K, 50 K, and 21 K fragments decreased by about 50%. The molecular weight of the new band was lower than that of the cross-linked product of 26 K, 50 K, and 21 K fragments, and higher than that of the dimer of actin. Based on this and some other results, we suggest that this band represented a cross-linked product of actin and the 50 K fragment. We also suggest that the decrease in the amount of cross-linked product of 26 K, 50 K, and 21 K fragments reflected the conformational change in subfragment-1 due to the binding of actin.     

Opposite effects of cofilin and profilin from porcine brain on rate of exchange of actin-bound adenosine 5'-triphosphate

Cofilin, an actin-binding protein isolated from porcine brain that reacts with actin in a 1:1 molar ratio [Nishida, E., Maekawa, S., & Sakai, H. (1984) Biochemistry 23, 5307-5313], decreases the rate of exchange of ATP bound to G-actin with 1,N6-ethenoadenosine 5'-triphosphate in solution. From analyses of the dependence of the exchange rate on the cofilin concentration under different KCl concentrations, dissociation constants (KD) for the cofilin-actin binding at 0, 50, and 140 mM KCl were determined to be 0.12, 0.15, and 0.25 microM, respectively. In contrast to cofilin, profilin isolated from porcine brain increases the rate of exchange of G-actin-bound ATP, like Acanthamoeba profilin. The kinetic analyses gave KD values for the profilin-actin binding of 1.1 and 1.5 microM, respectively, at 50 and 200 mM KCl.     

Polymeric C-terminal cross-linked material from type-I collagen. A modified method for purification, anomalous behaviour on gel filtration, molecular weight estimation, carbohydrate content and lipid content.

Polymeric cross-linked C-terminal peptide material (poly-alpha 1CB6) from mature bovine tendon type-I collagen was prepared and purified by a modification of the method previously described [Light & Bailey (1980) Biochem. J. 185, 373-381]. Poly-alpha 1CB6 was shown to exhibit concentration-dependent aggregation effects on gel filtration due to interaction with a filtration medium. The material had an amino acid content that was very similar to a mixture of alpha 1CB6 and alpha 1CB5. The material was shown to be polydisperse with a mol.wt. range of 50 000-350 000, but chromatographic fractions were relatively homogeneous over this molecular weight range with respect to amino-acid composition. The heterogeneity of the material was not due to incomplete CNBr peptide cleavage, as poly-alpha 1CB6 did not contain detectable quantities of methionine. The material showed no discrete bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis but gave a constant blue stain throughout the molecular weight range described above. Lipid analysis showed that the partially purified material contained elevated levels of stearate when compared to the crude CNBr-digested starting material. This may indicate the specific association of a stearic-acid-rich lipid with the peptide material. On carbohydrate analysis poly-alpha 1CB6 was shown to contain only galactose and glucose at levels of 0.72 and 0.28% respectively. The carbohydrate and amino acid analyses indicated that (alpha 1CB6)2-(alpha 1CB5)1 may be the basic cross-linked structural unit of poly-alpha 1CB6)2-(alpha 1CB5)1 units, although the carbohydrate analysis indicated that the higher molecular weight oligomers may be enriched in alpha 1CB6.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

Acanthamoeba actin and profilin can be cross-linked between glutamic acid 364 of actin and lysine 115 of profilin

Acanthamoeba profilin was cross-linked to actin via a zero-length isopeptide bond using carbodiimide. The covalently linked 1:1 complex was purified and treated with cyanogen bromide. This cleaves actin into small cyanogen bromide (CNBr) peptides and leaves the profilin intact owing to its lack of methionine. Profilin with one covalently attached actin CNBr peptide was purified by gel filtration followed by gel electrophoresis and electroblotting on polybase-coated glass-fiber membranes. Since the NH2 terminus of profilin is blocked, Edman degradation gave only the sequence of the conjugated actin CNBr fragment beginning with Trp-356. The profilin-actin CNBr peptide conjugate was digested further with trypsin and the cross-linked peptide identified by comparison with the tryptic peptide pattern obtained from carbodiimide-treated profilin. Amino-acid sequence analysis of the cross-linked tryptic peptides produced two residues at each cycle. Their order corresponds to actin starting at Trp-356 and profilin starting at Ala-94. From the absence of the phenylthiohydantoin-amino acid residues in specific cycles, we conclude that actin Glu-364 is linked to Lys-115 in profilin. Experiments with the isoforms of profilin I and profilin II gave identical results. The cross-linked region in profilin is homologous with sequences in the larger actin filament capping proteins fragmin and gelsolin.     

Cross-linking of actin to myosin subfragment 1: course of reaction and stoichiometry of products

The cross-linking of actin to myosin subfragment 1 (S-1) with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide was reexamined by using two cross-linking procedures [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306; Sutoh, K. (1983) Biochemistry 22, 1579-1585] and two independent methods for quantitating the reaction products. In the first approach, the cross-linked acto-S-1 complexes were cleaved with elastase at the 25K/50K and 50K/22K junctions in S-1. This enabled direct measurements of the cross-linked and un-cross-linked fractions of the 50K and 22K fragments of S-1. We found that in all cases actin was preferentially cross-linked to the 22K fragment and that the overall stoichiometry of the main cross-linked products was that of a 1:1 complex of actin and S-1. In the second approach, actin was cross-linked to tryptically cleaved S-1, and the course of these reactions was monitored by measuring the decay of the free 50K and 20K fragments and the formation of cross-linked products. After selecting the optimal cross-linking procedure and conditions, we determined that the rate of actin cross-linking to the 20K fragment of S-1 was 3-fold faster than the reaction with the 50K peptide. The overall rate of cross-linking actin to S-1 corresponded to the sum of the individual reactions of the 50K and 20K fragments, indicating their mutually exclusive cross-linking to actin. Thus, the reactions with tryptically cleaved S-1 were consistent with the 1:1 stoichiometry of actin and S-1 in the main cross-linked products and verified the preferential cross-linking of actin to the 20K fragment of S-1. These results are discussed in the context of the binding of actin to S-1.     

Isolation of low molecular weight actin-binding proteins from porcine brain

Three new actin-binding proteins having molecular weights of 26,000, 21,000, and 19,000 were isolated from porcine brain by DNase I affinity column chromatography. These proteins were released from the DNase I column by elution with a solution of high ionic strength. They were further purified by column chromatographies using hydroxyapatite, phosphocellulose, and Sephadex G-75. All of these actin-binding proteins behaved as monomeric particles in the gel filtration chromatography. After elution of the three actin-binding proteins, actin and profilin were recovered from the DNase I column with 2 M urea solution. The eluted was further purified by a cycle of polymerization and depolymerization and finally by gel filtration. Little difference in polymerizability was detected between the purified brain actin and muscle actin. After sedimentation of the polymerized brain actin, profilin was purified by DEAE-cellulose and gel filtration column chromatographies. In the assay of the action of these actin-binding proteins, the 26K protein was found to cause a large decrease in the rate of actin polymerization, while showing little effect on the extent of polymerization. The 21K protein decreased the steady-state viscosity of actin solution in a concentration-dependent manner irrespective of whether it was added before or after actin polymerization. It reacted with actin at a 1:1 molar ratio.