STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti-chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea-polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ-nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi , 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.     

Studies on muscle differentiation. IV. Electrophoretic and immunochemical analyses of tropomyosin from adult and embryonic skeletal muscles

Tropomyosin preparations from skeletal muscles of the adult frog, chick and rabbit were resolved in 8 M urea-polyacrylamide gel electrophoresis into at least 5 to 6 components. Of these, main components clearly reacted with anti-frog tropomyosin antiserum in agar diffusion test. Especially, main components of the frog tropomyosin preparation contained both genus- and organ-specific and genus- and organ-nonspecific antigens without being differentiated into separate entities. The chick tropomyosin preparation formed a single band when thioglycolic acid was included in 8 M urea-polyacrylamide gel electrophoresis. This single component was revealed in an SDS-polyacrylamide gel electrophoresis to be monomeric tropomyosin subunit with a molecular weight of 34,000. Both adult and embryonic chick tropomyosin preparations in their course of purification were observed in 8 M urea-polyacrylamide gel electrophoresis to decrease in amount of the monomeric component with a concomitant increase in number and in amount of polymerized components. It was concluded that the monomeric subunit was the major form of tropomyosin molecules in both adult and embryonic skeletal muscle extracts of the chick and that the polymerized components with inter-subunit disulfide bonds were formed in the course of purification of the preparations.     

Existence of common antigenic sites in tropomyosins.

1. Tropomyosins were extracted from vertebrate and invertebrate muscles, and their immunolo;ical characteristics were compared using antisera against tropomyosins from chicken skeletal and cardiac muscles. 2. Antigenic sites common to those of chicken skeletal muscle tropomyosin were found in all the tropomyosins tested, although the reactions of these common antigenic sites in an immunodiffusion test were weak in tropomyosins from phylogenetically distant animals. 3. An immunological difference was found between alpha-tropomyosins from chicken cardiac muscle and rabbit cardiac muscle. Thus they had specific antigenic sites in addition to the common ones. 4. A component was found in a 1 M KCL extract of Tetrahymena pyriformis which reacted with antiserum against chicken skeletal muscle tropomyosin.     

STUDIES ON MUSCLE DIFFERENTIATION. V. ANTIGENICITIES OF MYOSIN AND OF ACTIN FROM FROG SKELETAL MUSCLES1

Both intact and denatured preparations of myosin and actin from frog skeletal muscles produced in rabbits antisera containing antibodies against authentic myosin and actin, respectively, though being contaminated with antibodies against other proteins. Antigenicity of our frog myosin as revealed in agar diffusion tests was indistinguishable from that of cardiac muscle myosin from the same species. Similarly, skeletal muscle myosins from other amphibians shared to a certain extent immunological characteristics with our frog myosin, but those from avian and mammalian materials did not. Similarity in antigenicity was also demonstrated among our skeletal muscle actin, cardiac muscle actin from the same species and skeletal muscle actin from the other anurans studied. However, skeletal muscle actin from an urodele could not clearly be correlated in its immunological properties with our frog actin, and those from avian and mammalian materials were antigenically different from our frog actin. Thus, the degree of antigenic similarity of these muscle proteins seemed to be correlated with the phylogenic relationship of the animals so far studied. The results also indicated that our antisera could only be applied to immuno-cytological and immuno-embryological studies of myosin and actin when the antisera absorbed with the corresponding antigen preparations were used as negative controls.     

Coexistence of cardiac-type and fast skeletal-type myosin light chains in embryonic chicken cardiac muscle

Myosin from embryonic chicken ventricle contained a light chain component which comigrated with fast skeletal myosin light chain 1 (Lf1) on two dimensional electrophoresis in addition to cardiac type light chains (Lc1 and Lc2). Immunoblot analysis showed that this minor light chain band reacted with anti-Lf1 antibody. Antigens binding with anti-Lc1 and anti-Lf1 antibodies were located on myofibrils in embryonic cardiac muscle cells in vivo and in vitro. From these observations, we conclude that a small amount of Lf1 exists in embryonic chicken cardiac muscle.     

Differentiation of troponin in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy

The differentiation of troponin (TN) in cardiac and skeletal muscles of chicken embryos was studied by indirect immunofluorescence microscopy. Serial sections of embryos were stained with antibodies specific to TN components (TN-T, -I, and -C) from adult chicken cardiac and skeletal muscles. Cardiac muscle began to be stained with antibodies raised against cardiac TN components in embryos after stage 10 (Hamburger and Hamilton numbering, 1951, J. Morphol. 88:49-92). It reacted also with antiskeletal TN-I from stage 10 to hatching. Skeletal muscle was stained with antibodies raised against skeletal TN components after stage 14. It also reacted with anticardiac TN-T and C from stage C from stage 14 to hatching. It is concluded that, during embryonic development, cardiac muscle synthesizes TN-T and C that possess cardiac- type antigenicity and TN-I that has antigenic determinants similar to those present in cardiac as well as in skeletal muscles. Embryonic skeletal muscle synthesizes TN-I that possesses antigenicity for skeletal muscle and TN-T and C which share the antigenicities for both cardiac and skeletal muscles. Thus, in the development of cardiac and skeletal muscles, a process occurs in which the fiber changes its genomic programming: it ceases synthesis of the TN components that are immunologically indistinguishable from one another and synthesizes only tissue-type specific proteins after hatching.     

Immunochemical evidence for the species-specificity of mammalian cardiac myosin and heavy meromyosin.

Structural differences between various myosins were investigated by means of antibodies to heavy meromyosin, a tryptic subfragment of myosin. Heavy meromyosin was purified from rabbit white skeletal and from pig and human cardiac muscles by gel filtration, and antisera were produced in guinea pigs. Analyses, carried out with the quantitative micro-complement fixation technique, indicated that the antibodies were specific to heavy meromyosin and myosin and not to other contractile proteins. For each muscle type, the corresponding intact myosin reacted, and the degree of dixation was always lower than with heavy meromyosin (50 and 70% fixation respectively). This vertical shift was the same for the three muscle types, indicating that the heavy meromyosin represent corresponding fragments of the myosin molecule from one muscle to the other. Antisera to pig or human cardiac heavy meromyosin clearly distinguished antigens (heavy meromyosins, myosins, or crude extracts) from the ventricles of various heterologous species. Relative to pig, the immunological distances were 50 for the rabbit, 73 for the rat and greater than 100 for human and mice. Relative to human, these values were 20 for the rat, 60 for the rabbit, 72 for the pig. These data provide direct evidence that mammalian cardiac myosin is species-specific.     

Visualization of monoclonal antibody binding to tropomyosin on native smooth muscle thin filaments by electron microscopy

We have used three different monoclonal antibodies (LCK16, JLH2 and JLF15) to tropomyosin for the localization of tropomyosin molecules within smooth muscle thin filaments. Thin filaments were incubated with monoclonal antibodies and visualized by negative staining electron microscopy. All three monoclonal antibodies caused the aggregation of thin filaments into ordered bundles, which displayed cross-striations with a periodicity of 37 ± 1 nm. In contrast, conventional rabbit antiserum to tropomyosin distorted and aggregated the thin filaments without generating cross-striations. Therefore, monoclonal antibodies to tropomyosin allow us, for the first time, to observe directly the distribution of tropomyosin molecules along the thin filaments of smooth muscle cells. The binding sites of the antibodies to skeletal muscle tropomyosin were examined by decorating tropomyosin paracrystals with monoclonal antibodies. The LCK16 monoclonal antibody binds the narrow band of tropomyosin paracrystals, whereas the JLF15 antibody binds the wide band of tropomyosin paracrystals.     

Changes in tropomyosin subunits and myosin light chains during development of chicken and rabbit striated muscles.

We have selected tropomyosin subunits and myosin light chains as representative markers of the myofibrillar proteins of the thin and thick filaments and have studied changes in the type of proteins present during development in chicken and rabbit striated muscles. The β subunit of tropomyosin is the major species found in all embryonic skeletal muscles studied. During development the proportion of the α subunit of tropomyosin gradually increases so that in adult skeletal muscles the α subunit is either the only or the major species present. In contrast, cardiac muscles of both chicken and rabbit contain only the α subunit which remains invariant with development. Two subspecies of the α subunit of tropomyosin which differ in charge only were found in adult and embryonic chicken skeletal muscles. Only one of these subspecies seems to be common to chicken cardiac tropomyosin. With respect to myosin light chains, embryonic skeletal fast muscle myosin of both species resembles the adult fast muscle myosin except that the LC₃ light chain characteristic of the adult skeletal fast muscle is present in smaller amounts. The significance of these isozymic changes in the two myofibrillar proteins is discussed in terms of a model of differential gene expression during development of chicken and rabbit skeletal muscles.     

Subcellular localization of glycogen synthase with monoclonal antibodies

Two monoclonal antibodies, designated 7H5 and 8E11, were produced against glycogen synthase purified from rabbit skeletal muscle. Both antibodies were of the IgG1 (k) isotype. Western blot analysis of extracts of rat and rabbit tissues showed that antibody 7H5 recognized glycogen synthase from skeletal and cardiac muscles, but not from liver. Antibody 8E11 gave similar results but the responses were weaker. Antibody 7H5 also recognized a 69,000 dalton tryptic fragment of glycogen synthase whereas antibody 8E11 did not bind this fragment. Immunocytochemical staining of rabbit skeletal muscle with antibody 7H5 indicated two major sites of glycogen synthase localization. A granular localization present in the cytoplasm and a band-like staining associated with the Z-disk region of the myofibrils. Rabbit cardiac muscle presented a similar pattern though less cytoplasmic staining was apparent. An assay of subcellular fractions for glycogen synthase indicated that the enzyme in cardiac and skeletal muscles is distributed between the soluble (80-90%) and myofibrilar (10-20%) fractions of the tissues. These results provide direct evidence for the presence of glycogen synthase in subcellular fractions other than the soluble fraction of skeletal and cardiac muscles.     

A study of anti-group A streptococcal monoclonal antibodies cross-reactive with myosin

Anti-group A streptococcal monoclonal antibodies were obtained from BALB c/BYJ mice immunized with purified membranes from M type 5 Streptococcus pyogenes. Two of the anti-streptococcal monoclonal antibodies were previously shown to cross-react with muscle myosin. In this study the monoclonal antibodies were reacted with tissue sections of normal human heart and skeletal muscle. Antibody binding was estimated by indirect immunofluorescence and immunoperoxidase techniques. Both of the monoclonal antibodies (36.2.2 and 54.2.8) investigated in this report reacted with heart and/or skeletal muscle sections. When evaluated by immunofluorescence, monoclonal antibody 54.2.8 demarcated the periphery of cardiac striated muscle cells and reacted to a lesser degree with subsarcolemmal components. Monoclonal antibody 36.2.2 failed to react with heart sections, but both of the monoclonal antibodies reacted strongly with skeletal muscle sections. Results similar to those observed with indirect immunofluorescence were obtained with the immunoperoxidase technique. By Western immunoblotting and competitive inhibition assays, monoclonal antibodies 36.2.2 and 54.2.8 both were found to react with the heavy chain of skeletal muscle myosin. However, only 54.2.8 reacted with the heavy chain of cardiac myosin. The specificity of the monoclonal antibodies for subfragments of skeletal muscle myosin indicated that monoclonal antibody 36.2.2 was specific for light meromyosin fragments, whereas 54.2.8 reacted with both heavy and light meromyosin. The data demonstrated that two monoclonal antibodies against streptococci were specific for skeletal muscle and/or cardiac myosin and for subfragments of the myosin molecule. The reactions of the monoclonal antibodies with human tissue sections were consistent with the immunochemical reactions of the monoclonal antibodies with both denatured and native myosin.     

Tissue specificity of tropomyosin from the crayfish, Cambarus clarki

The molecular heterogeneity and tissue specificity of crustacean tropomyosin were investigated, using muscle and nonmuscle tissues from the crayfish, Cambarus clarki. In muscle, three types of tropomyosin isoforms were found on two-dimensional gel electrophoresis. One of them was specific to cardiac muscle, and the other two were shared by skeletal and visceral muscles. In nonmuscle tissues, four types of isoforms were found on two-dimensional gel electrophoresis and in immunoreplica tests using an antiserum against crayfish skeletal muscle tropomyosin. Two of them were common to the muscle isoforms, but the other two were not detected in muscles. Furthermore, nonmuscle tissues contained several peculiar isoforms, the electrophoretic mobilities of which were considerably higher than those of the other isoforms mentioned above. When tropomyosin was purified from the mid-gut gland, these isoforms with high mobilities were found in the crude tropomyosin preparation. These results showed that the crayfish tropomyosin was heterogeneous and that the isoforms were distributed in a tissue-specific manner, like vertebrate tropomyosin. However, the results did not coincide with those of our previous study on horseshoe crab tropomyosin, which showed molecular heterogeneity but no tissue specificity. In view of the difference in the isoform distributions between the two major groups (Crustacea and Merostomata) of Arthropoda, the significance of the tissue specificity of tropomyosin isoforms was discussed.     

Developmental changes of cardiac and slow skeletal muscle troponin T expression in chicken cardiac and skeletal muscles

Numerous troponin T (TnT) isoforms are produced by alternative splicing from three genes characteristic of cardiac, fast skeletal, and slow skeletal muscles. Apart from the developmental transition of fast skeletal muscle TnT isoforms, switching of TnT expression during muscle development is poorly understood. In this study, we investigated precisely and comprehensively developmental changes in chicken cardiac and slow skeletal muscle TnT isoforms by two-dimensional gel electrophoresis and immunoblotting with specific antisera. Four major isoforms composed of two each of higher and lower molecular weights were found in cardiac TnT (cTnT). Expression of cTnT changed from high- to low-molecular-weight isoforms during cardiac muscle development. On the other hand, such a transition was not found and only high-molecular-weight isoforms were expressed in the early stages of chicken skeletal muscle development. Two major and three minor isoforms of slow skeletal muscle TnT (sTnT), three of which were newly found in this study, were expressed in chicken skeletal muscles. The major sTnT isoforms were commonly detected throughout development in slow and mixed skeletal muscles, and at developmental stages until hatching-out in fast skeletal muscles. The expression of minor sTnT isoforms varied from muscle to muscle and during development.     

Heterogeneity and distribution of fast myosin heavy chains in some adult vertebrate skeletal muscles.

Three monoclonal antibodies, LM5, F2 and F39 raised to chicken fast skeletal muscle myosin, specific for myosin heavy chain (MHC) subunit, were used to study the composition and distribution of this protein in some vertebrate skeletal muscles. These antibodies in immunohistochemical investigations did not react with the majority of the type I fibres in most muscles. Antibodies LM5 and F39 stained all the type II fibres in all the adult chicken skeletal muscles studied. Antibody F2 also stained all the type II fibres in most chicken skeletal muscles tested except in gastrocnemius in which a proportion of both the type IIA and IIB fibres either did not stain or stained only weakly. Antibody F2 unlike LM5 and F39 stained most of the type IIIB fibres in anterior latissimus dorsi (ALD) and IB fibres in red strip of chicken Pectoralis muscle. Antibodies LM5 and F2 in the rat diaphragm reacted with all the type IIA and IIB fibres, while antibody F39 stained only the type IIB fibres darkly with most IIA fibres being either not stained or only weakly stained. In the rat extensor digitorum longus (EDL) and tibialis anterior (TA) muscles, antibody LM5 stained all the IIA and IIB fibres. Antibody F2 in these muscles stained all the type IIA fibres but only a proportion of the IIB fibres. The remaining IIB fibres were either unstained or only weakly positive. Antibody F39 in rat EDL and TA muscles did not only distinguish subgroups of IIB fibres (dark, intermediate and negative or very weak) but also of the IIA fibres. These three antibodies used together therefore detected a great deal of heterogeneity in the myosin heavy chain composition and muscle fibre types of several skeletal muscles.     

Structural role of tropomyosin in muscle regulation: analysis of the x-ray diffraction patterns from relaxed and contracting muscles

Previous work has shown that there are significant differences in the X-ray diffraction patterns obtained from relaxed and contracting muscles. We show that some of these changes can be explained in terms of a small movement (~ 5 to 15 Å) of the tropomyosin molecules in the groove of the actin helix. The position of the tropomyosin in relaxed skeletal muscle is such that it might physically block or at least structurally alter the cross-bridge attachment site on actin, whereas in contracting skeletal muscle the tropomyosin moves to a position well clear of the attachment site. The movement of the tropomyosin molecules is apparently smaller in molluscan muscles during tonic contraction than in vertebrate skeletal muscle. We suggest a possible relationship between the smaller movement of the tropomyosin and the “catch” response of molluscan muscles.We also show that any increase of intensity on the 59 Å and 51 Å layer-lines is most likely to be associated with some extra mass (HMM S-1) attaching to the actin molecules. Such a change cannot be explained in terms of a change in tropomyosin structure or in the order within the thin filaments. Since changes on these two layer-lines have been observed during contraction, this provides good evidence for cross-bridge attachment to actin in contracting muscles.     

Troponin of asynchronous flight muscle

Troponin has been prepared from the asynchronous flight muscle of Lethocerus (water bug) taking special care to prevent proteolysis. The regulatory complex contained tropomyosin and troponin components. The troponin components were Tn-C (18,000 Mr), Tn-T (apparent Mr 53,000) and a heavy component, Tn-H (apparent Mr 80,000). The troponin was tightly bound to tropomyosin and could not be dissociated from it in non-denaturing conditions. A complex of Tn-T, Tn-H and tropomyosin inhibited actomyosin ATPase activity and the inhibition was relieved by Tn-C from vertebrate striated muscle in the presence of Ca2+. However, unlike vertebrate Tn-I, Tn-H by itself was not inhibitory. Monoclonal antibodies were obtained to Tn-T and Tn-H. Antibody to Tn-T was used to screen an expression library of Drosophila cDNA cloned in lambda phage. The sequence of cDNA coding for the protein was determined and hence the amino acid sequence. The Drosophila protein has a sequence similar to that of vertebrate skeletal and cardiac Tn-T. The sequence extends beyond the carboxyl end of the vertebrate sequences, and the last 40 residues are acidic. Part of the sequence of Drosophila Tn-T is homologous to the carboxyl end of the Drosophila myosin light chain MLC-2 and one anti-Tn-T antibody cross-reacted with the light chain. Lethocerus Tn-H is related to the large tropomyosins of Drosophila flight muscle, for which the amino acid sequence is known, since antibodies that recognize this component also recognize the large tropomyosins. Tn-H is easily digested by calpain, suggesting that part of the molecule has an extended configuration. Electron micrographs of negatively stained specimens showed that Lethocerus thin filaments have projections at about 39 nm intervals, which are not seen on thin filaments from vertebrate striated muscle and are probably due to the relatively large troponin complex. Decoration of the thin filaments with myosin subfragment-1 in rigor conditions appeared not to be affected by the troponin. The troponin of asynchronous flight muscle lacks the Tn-I component of vertebrate striated muscle. Tn-H occurs only in the flight muscle and may be involved in the activation of this muscle by stretch.     

Tropomyosin from the striated muscles of carp (Cyprinus carpio) and of icefish (Channichthys rhinoceratus).

Tropomyosin of fast-twitch, slow-twitch and cardiac muscles of carp and icefish has been isolated by hydroxyapatite chromatography. The subunit distribution has been investigated by polyacrylamide gel electrophoresis and by peptide mapping. The purified skeletal muscle tropomyosins all belong to the alpha family and differ from higher vertebrate tropomyosin by the lack of beta subunits. Specific alpha isotypes are however encountered in fast-twitch fibres (alpha w subunit) and slow-twitch or intermediate (pink) fibres (alpha and alpha w subunits). The amino acid compositions and the paracrystals formed by the carp alpha w alpha w and alpha alpha w tropomyosins do not differ markedly from that of rabbit alpha alpha chains. They differ however by their capability to inhibit the ATPase activity of rabbit skeletal muscle acto-HMM system. A beta-like subunit is found in carp cardiac tropomyosin, in the proportion of 25% of the native protein, but not in icefish heart.     

Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.     

Preparation and properties of vertebrate smooth-muscle myofibrils and actomyosin.

A new technique for obtaining a myofibril-like preparation from vertebrate smooth muscle has been developed. An actomyosin can be readily extracted from these myofibrils at low ionic strength and in yields 20 times as high as previously reported. The protein composition of all preparations has been monitored using dodecylsulfate-gel electrophoresis. By this method smooth muscle actomyosin showed primarily only the major proteins, myosin, actin and tropomyosin, while the myofibrils contained, additionally, three new proteins not previously described with polypeptide chain weights of 60000, 110000 and 130000. The ATPase activities of both the myofibrils and actomyosin preparations are considerably higher than previously described for vertebrate smooth muscle. They are sensitive to micromolar Ca2+ ion concentrations to the same degree as comparable skeletal and cardiac muscle preparations, even though troponin-like proteins could not be identified in these smooth muscle preparations. From the latter observation and the presence of Ca2+-sensitivity in tropomyosin-free actomyosin it is suggested that this calcium sensitivity is, as in some invertebrate muscles, a property of the myosin molecule.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)