Reiji Natori, Setsuro Ebashi, and excitation-contraction coupling

The achievements of Natori and Ebashi, which greatly contributed to the progress in studies of excitation–contraction coupling, were reviewed. Natori succeeded in removing the cell membrane of an isolated fiber of skeletal muscle to prepare a skinned fiber, which still responded to an electrical stimulation with propagated contraction. Skinned fibers showed elastic extensibility beyond the elastic limit of intact muscle fibers. Based on this elasticity Natori predicted the presence of an elastic components, later found as connectin. Skinned fibers, an excellent experimental system, contributed greatly to the progress in subsequent studies. Ebashi showed that the essential principle of the relaxing factor was not the ATP-regenerating enzymes as generally thought, but a particulate fraction with MgATPase. Then he clearly showed that a minute amount of Ca²⁺ is necessary for the contractile reaction of actomyosin, and that the relaxing factor strongly accumulates Ca²⁺ in the presence of ATP and causes relaxation by the removal of Ca²⁺. He further discovered that the Ca²⁺-induced regulation of the contractile reaction of the myosin–actin system requires the presence of tropomyosin and a new protein, troponin. Troponin binds to a specific site on tropomyosin, which in turn binds to actin in the thin filament. Troponin is the Ca²⁺-receptive protein, and changes in troponin molecules upon Ca²⁺ binding is transmitted to actin through tropomyosin to regulate the actin–myosin interaction. Through these findings, the excitation was connected by Ca²⁺ with the contraction.     

Calcium ion regulation of muscle contraction: The regulatory role of troponin T

The relaxation and contraction in vertebrate skeletal muscle are regulated by Ca2+ through troponin and tropomyosin, which are located in the thin filament. Troponin is composed of three components, troponins C, I and T. In this review article, the Ca2+-regulatory mechanism is discussed with particular reference to the regulatory properties of troponin T.     

Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component,Troponin T

The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca²⁺ sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca²⁺ activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca²⁺ binding measurements. Ca²⁺ activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca²⁺ sensitivity observed in Ca²⁺ binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.     

Structural basis for the regulation of muscle contraction by troponin and tropomyosin

The molecular switching mechanism governing skeletal and cardiac muscle contraction couples the binding of Ca²⁺ on troponin to the movement of tropomyosin on actin filaments. Despite years of investigation, this mechanism remains unclear because it has not yet been possible to directly assess the structural influence of troponin on tropomyosin that causes actin filaments, and hence myosin-crossbridge cycling and contraction, to switch on and off. A C-terminal domain of troponin I is thought to be intimately involved in inducing tropomyosin movement to an inhibitory position that blocks myosin-crossbridge interaction. Release of this regulatory, latching domain from actin after Ca²⁺ binding to TnC (the Ca²⁺ sensor of troponin that relieves inhibition) presumably allows tropomyosin movement away from the inhibitory position on actin, thus initiating contraction. However, the structural interactions of the regulatory domain of TnI (the “inhibitory” subunit of troponin) with tropomyosin and actin that cause tropomyosin movement are unknown, and thus, the regulatory process is not well defined. Here, thin filaments were labeled with an engineered construct representing C-terminal TnI, and then, 3D electron microscopy was used to resolve where troponin is anchored on actin-tropomyosin. Electron microscopy reconstruction showed how TnI binding to both actin and tropomyosin at low Ca²⁺ competes with tropomyosin for a common site on actin and drives tropomyosin movement to a constrained, relaxing position to inhibit myosin-crossbridge association. Thus, the observations reported reveal the structural mechanism responsible for troponin-tropomyosin-mediated steric interference of actin-myosin interaction that regulates muscle contraction.     

Functional effects of substitutions I92T and V95A in actin-binding period 3 of tropomyosin

Tropomyosin polymerizes along actin filaments and together with troponin regulates muscle contraction in a Ca-dependent manner. Actin-binding periods are homologous residues, which repeat along tropomyosin sequence, form tropomyosin-actin interface and determine regulatory functions. To learn how period 3 is involved in tropomyosin functions we examined effects of two mutations in Tpm1.1, I92T and V95A, which have been linked to dilated and hypertrophic cardiomyopathies characterized respectively by hyper- and hypocontractile phenotypes. In this work the functional consequences of both mutations were studied in vitro by using actin thin filaments reconstituted in the presence of mutant Tpm1.1 homodimers carrying the substitutions in both tropomyosin chains, Tpm1.1 heterodimers with substitution only in one Tpm1.1 chain, and Tpm1.1/Tpm2.2 heterodimers with substitution in Tpm1.1 chain and wild type Tpm2.2 in the second chain. The presence of the substitution I92T decreased the tropomyosin affinity for actin, abolished Ca²⁺-dependent activation of the actomyosin ATPase, decreased the sensitivity of the tropomyosin-troponin complex to subsaturating Ca²⁺ concentrations and reduced the cooperativity of the myosin-induced transition of the thin filament to a fully active state. The substitution V95A had opposite effects: increased actin affinity, increased the actomyosin ATPase activity above the level observed for wild type Tpm and increased cooperativity of myosin-induced activation of the thin filaments reconstructed with homo- and heterodimers of tropomyosin. Substitutions I92T and V95A were dominant, but the formation of heterodimers modified the effects observed for homodimers.     

Preserved Close Linkage Between the Genes Encoding Troponin I and Troponin T, Reflecting an Evolution of Adapter Proteins Coupling the Ca2+ Signaling of Contractility

Ca²⁺-regulated motility is essential to numerous cellular functions, including muscle contraction. Systems with troponin C, myosin light chain, or calmodulin as the Ca²⁺ receptor have evolved in striated muscle and other types of cells to transduce the cytoplasm Ca²⁺ signals into allosteric conformational changes of contractile proteins. While these Ca²⁺ receptors are homologous proteins, their coupling to the responding elements is quite different in various cell types. The Ca²⁺ regulatory system in vertebrate striated muscle represents a highly specialized such signal transduction pathway consisting of the troponin complex and tropomyosin associated with the actin filament. To understand the molecular mechanism in the Ca²⁺ regulation of muscle contraction and cell motility, we have revealed a preserved ancestral close linkage between the genes encoding two of the troponin subunits, troponin I and troponin T, in the genome of mouse. The data suggest that the troponin I and troponin T genes may have originated from a single locus and evolved in parallel to encode a striated muscle-specific adapter to couple the Ca²⁺ receptor, troponin C, to the actin–myosin contractile machinery. This hypothesis views the three troponin subunits as two structure–function domains: the Ca²⁺ receptor and the signal transducing adapter. This model may help to further our understanding of the Ca²⁺ regulation of muscle contraction and the structure–function relationship of other potential adapter proteins which are converged to constitute the Ca²⁺ signal transduction pathways governing nonmuscle cell motility. Received: 15 April 1999 / Accepted: 15 July 1999     

大鼠骨骼肌快肌肌钙蛋白T的同工型

骨骼肌快肌的收缩主要是由钙离子通过肌钙蛋白所调节控制。这些肌钙蛋白位于肌纤维之中。肌蛋白包括肌钙蛋白T、肌钙蛋白C、肌钙蛋白I。采用双向聚丙烯酰胺凝胶电泳和免疫学技术,对大鼠胚胎、新生大鼠和成年大鼠的骨能肌快肌肌钙蛋白T的同工型进行了研究。在成年大鼠的骨能肌快肌中,发现了10种肌钙蛋白T同工型。在大鼠胚胎和新生大鼠的骨能肌中,发现了7种肌钙蛋白T同工型。作为不同动物、不同发育阶段和不同组织发育的特殊标记,这些肌钙蛋白T同工型具有重要意义[动物学报49(3)：362—369,2003]。     

Calcium sensitive binding of troponin to actin-tropomyosin: a two-site model for troponin action

Troponin reconstituted from the inhibitory component (troponin-I) and calcium binding protein (troponin-C) binds readily to actin-tropomyosin in 0.1 mm-EGTA but only poorly in 0.01 mm-CaCl₂ or 0.1 mm-Ca-EGTA. Troponin prepared by extraction of myofibrils with mersalyl, an organic mercurial, contains only these two components and also shows this calcium-sensitive binding and is deficient in its ability to bind to tropomyosin. Troponin-I + C is unable to confer calcium sensitivity on the Mg²⁺ activated actomyosin ATPase in concentrations at which native troponin is fully effective and the ATPase activity remains high in the absence of calcium. Addition of the tropomyosin binding component (troponin-T) to the other two components restores their ability to remain associated with actin-tropomyosin in the presence of calcium as does native troponin; calcium sensitivity is also regained. The results of these experiments have been interpreted in terms of a two-site mechanism of troponin action.     

Background to the discovery of troponin and Setsuro Ebashi's contribution to our knowledge of the mechanism of relaxation in striated muscle

The discovery of the actomyosin system provided for the first time a model system that enabled the study of the role of the muscle protein components in the contraction and relaxation cycle to be undertaken. It soon became apparent that ATP was essential for both processes but progress really began when it became clear that components both in the myofibrillar and sarcoplasmic fractions were involved in relaxation. After it was apparent that a trace of calcium was required for the activation of the MgATPase of the myofibrils it was shown that an active calcium pump was located in the sarcoplasmic reticulum. The report by Ebashi in 1963 that a new myofibrillar protein, troponin, was the target for calcium opened up the investigation of the calcium control of the MgATPase. Troponin was shown to be a complex of troponin C, I and T, each protein being under individual genetic control and existing in isoforms specific for the muscle type. The unique forms of troponin I and T in cardiac muscle make them the biomarkers of choice for cardiac injury.     

Molecular structure of troponin C from chicken skeletal muscle at 3Å resolution

Troponin C is the Ca²⁺-binding subunit of the troponin complex and is involved in the calcium control of muscle contraction. The X-ray structure of chicken TnC has been determined at 3Å resolution using a single heavy atom derivative and application of a novel phase improvement and phase extension procedure. The protein has an unusual dumbbell-shape with a length of about 70A. The N- and C-domains are connected by a single long α-helix of about 9 turns. Two metal binding sites (the Ca²⁺-Mg²⁺ sites) in the C-domain are occupied by metal ions in the crystals and the helix-loop-helix Ca²⁺ -binding folds are very similar to those in other known Ca²⁺ -binding proteins. In contrast, the Ca²⁺ -specific sites in the N-domain appear unoccupied and the two putative Ca²⁺ -binding folds have a vastly different structural arrangement. The conformational rearrangements in the N-domain upon Ca²⁺ binding are believed to be the trigger for a cascade of protein-protein interaction alterations which lead to muscle contraction.     

Calcium ion and troponin: Professor S. Ebashi's epoch-making achievement

The processes by which Professor Setsuro Ebashi accomplished his great work are described. Independently of Marsh, Ebashi discovered the relaxing factor in homogenized muscle and showed that it has a lipid-containing particulate fraction with ATPase activity, later identified as the sarcoplasmic reticulum. He then solved the mechanism of relaxation of the relaxing factor through the following findings. A minute amount of calcium ion (Ca²⁺) is necessary for the physiological contractile reaction. The relaxing factor strongly accumulates Ca²⁺ in the presence of ATP and sufficiently removes Ca²⁺ from the contractile system to bring about relaxation. Ebashi found that the contractile reaction of myosin and actin is regulated by Ca²⁺ only in the presence of a tropomyosin-like protein factor, which he later showed to be a complex of tropomyosin and a new protein, troponin. He proved that troponin is the Ca²⁺-receptive protein and proposed the correct scheme for the molecular mechanism of regulation of contraction and relaxation.     

Interactions of desensitized actomyosin with tropomyosin, troponin A, troponin B, and polyanions

Troponin B is an inhibitor of the Mg⁺⁺-activated ATPase activity of actomyosin. The inhibitory effect, which is observed, however, depends upon whether tropomyosin is also present. In the absence of tropomyosin the inhibition by troponin B is markedly reduced by increasing the ionic strength from 0.03 to 0.07, but is not affected by calcium up to a concentration of 10^-4 M. Troponin A relieves the inhibition in both the absence and presence of calcium, an effect which is also shown by many polyanions and is illustrated by using RNA. Tropomyosin enhances the inhibitory effect of troponin B and renders it more resistant to increasing ionic strength but it does not make the inhibition calcium-sensitive. However, when troponin A or low concentrations of polyanions are added to troponin B and tropomyosin, the actomyosin ATPase activity becomes calcium-sensitive; i.e., in the presence of tropomyosin, troponin A or polyanions do not relieve the inhibitory action of troponin B in the absence of calcium but only in its presence. In marked contrast to this is the effect of troponin A in the absence of tropomyosin where it neutralizes the effect of troponin B under all conditions. Thus troponin A and the polyanions both confer calcium regulation on the troponin B-tropomyosin system. The similar effects exhibited by troponin A and the polyanions suggest that the addition of net negative charge to troponin B is an important factor in the conferral of calcium sensitivity. It is also clear that tropomyosin is an essential component of the regulatory mechanism.     

Troponin and its components from ascidian smooth muscle

Troponin was isolated from the thin filaments of ascidian smooth muscle and separated into three components by ion-exchange chromatography, the molecular weights of which were 33,000, 24,000, and 18,000, respectively. The three components were designated as troponin t (TN-T), troponin I (TN-I), and troponin C (TN-C) in order of molecular weight, since each component had properties similar to those of the respective components of vertebrate skeletal-muscle troponin. The ascidian troponin or the mixture of the three components conferred Ca2+-sensitivity on reconstituted rabbit actomyosin in the presence of tropomyosin. One of the characteristics of the ascidian troponin was Ca2+-dependent activation of actin-myosin interaction in collaboration with tropomyosin, whereas its inhibitory action on the actomyosin ATPase in the absence of Ca2+ was less remarkable. From this, it is concluded that in the ascidian smooth muscle actin-myosin interaction is regulated by an actin-linked troponin-tropomyosin system, but the ascidian troponin acts as a Ca2+-dependent activator of an actomyosin system.     

The relaxing protein system of striated muscle. Resolution of the troponin complex into inhibitory and calcium ion-sensitizing factors and their relationship to tropomyosin

1. A method involving isoelectric precipitation and chromatography on SE-Sephadex (sulphoethyl-Sephadex) is described for the preparation of the troponin complex free of tropomyosin from low-ionic-strength extracts of natural actomyosin and myofibrils. 2. Purified troponin complex required tropomyosin to inhibit the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin in the presence of ethanedioxybis(ethylamine)tetra-acetate. An upper limit of 35000 for the `molecular weight' of the troponin complex was derived from the amounts required to bring about 50% of the maximum inhibition of the Mg²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin of known concentration. 3. In the presence of dissociating reagents the troponin complex could be dissociated into inhibitory and Ca²⁺-sensitizing factors, which could be isolated separately on SE-Sephadex. The inhibitory factor inhibited the Mg²⁺-stimulated adenosine triphosphatase activity and superprecipitation of desensitized actomyosin independently of the concentration of free Ca²⁺ in the medium. 4. The Ca²⁺-sensitizing factor changed its electrophoretic mobility on polyacrylamide gel in the presence of ethanedioxybis(ethylamine)tetra-acetate. It formed a complex with the inhibitory factor at low ionic strength and the original biological activity of the troponin complex could be restored on mixing the inhibitory factor with the Ca²⁺-sensitizing factor in the ratio of about 3:2. 5. Evidence is presented indicating that the ability of tropomyosin preparations to restore relaxing-protein-system activity to the troponin complex and their inhibitory effect on the Ca²⁺-stimulated adenosine triphosphatase activity of desensitized actomyosin are two properties of different stability to preparative procedures and tryptic digestion. This suggests that the relaxing protein system of muscle may contain another as yet uncharacterized component.     

Troponin-like proteins from muscles of the scallop, Aequipecten irradians.

Ca2+ regulation of molluscan actomyosin adenosine triphosphatase is known to be associated with the myosin molecule. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, however, also suggests the possible presence of troponin, a thin-filament-linked Ca2+-regulatory complex. In the present study, scallop troponin and tropomyosin were prepared and complexed with rabbit actin; the resulting synthetic thin filaments form a Ca2+-dependent actomyosin adenosine triphosphatase with Ca2+-insensitive rabbit myosin, indicating that the troponin in scallops is potentially functional. Scallop troponin I was isolated and mixed with chicken troponin C and troponin T, forming a functional hybrid troponin complex, indicating that scallop and vertebrate troponins may act by a common mechanism. Densitometry of sodium dodecyl sulphate/polyacrylamide gels reveals that in synthetic thin filaments there are larger amounts of troponin than are present in native thin filaments. Amounts present in the intact muscle were not determined.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

Troponin Regulatory Function and Dynamics Revealed by H/D Exchange-Mass Spectrometry

Muscle contraction is tightly regulated by Ca²⁺ binding to the thin filament protein troponin. The mechanism of this regulation was investigated by detailed mapping of the dynamic properties of cardiac troponin using amide hydrogen exchange-mass spectrometry. Results were obtained in the presence of either saturation or non-saturation of the regulatory Ca²⁺ binding site in the NH₂ domain of subunit TnC. Troponin was found to be highly dynamic, with 60% of amides exchanging H for D within seconds of exposure to D₂O. In contrast, portions of the TnT-TnI coiled-coil exhibited high protection from exchange, despite 6 h in D₂O. The data indicate that the most stable portion of the trimeric troponin complex is the coiled-coil. Regulatory site Ca²⁺ binding altered dynamic properties (i.e. H/D exchange protection) locally, near the binding site and in the TnI switch helix that attaches to the Ca²⁺-saturated TnC NH₂ domain. More notably, Ca²⁺ also altered the dynamic properties of other parts of troponin: the TnI inhibitory peptide region that binds to actin, the TnT-TnI coiled-coil, and the TnC COOH domain that contains the regulatory Ca²⁺ sites in many invertebrate as opposed to vertebrate troponins. Mapping of these affected regions onto the troponin highly extended structure suggests that cardiac troponin switches between alternative sets of intramolecular interactions, similar to previous intermediate resolution x-ray data of skeletal muscle troponin.     

Significance of Troponin Dynamics for Ca2+-mediated Regulation of Contraction and Inherited Cardiomyopathy

Ca²⁺ dissociation from troponin causes cessation of muscle contraction by incompletely understood structural mechanisms. To investigate this process, regulatory site Ca²⁺ binding in the NH₂-lobe of subunit troponin C (TnC) was abolished by mutagenesis, and effects on cardiac troponin dynamics were mapped by hydrogen-deuterium exchange (HDX)-MS. The findings demonstrate the interrelationships among troponin''s detailed dynamics, troponin''s regulatory actions, and the pathogenesis of cardiomyopathy linked to troponin mutations. Ca²⁺ slowed HDX up to 2 orders of magnitude within the NH₂-lobe and the NH₂-lobe-associated TnI switch helix, implying that Ca²⁺ greatly stabilizes this troponin regulatory region. HDX of the TnI COOH terminus indicated that its known role in regulation involves a partially folded rather than unfolded structure in the absence of Ca²⁺ and actin. Ca²⁺-triggered stabilization extended beyond the known direct regulatory regions: to the start of the nearby TnI helix 1 and to the COOH terminus of the TnT-TnI coiled-coil. Ca²⁺ destabilized rather than stabilized specific TnI segments within the coiled-coil and destabilized a region not previously implicated in Ca²⁺-mediated regulation: the coiled-coil''s NH₂-terminal base plus the preceding TnI loop with which the base interacts. Cardiomyopathy-linked mutations clustered almost entirely within influentially dynamic regions of troponin, and many sites were Ca²⁺-sensitive. Overall, the findings demonstrate highly selective effects of regulatory site Ca²⁺, including opposite changes in protein dynamics at opposite ends of the troponin core domain. Ca²⁺ release triggers an intramolecular switching mechanism that propagates extensively within the extended troponin structure, suggests specific movements of the TnI inhibitory regions, and prominently involves troponin''s dynamic features.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Effect of phosphorylation of porcine cardiac troponin I by 3':5'-cyclic AMP-dependent protein kinase on the actomyosin ATPase activity

1. Porcine cardiac native tropomyosin was phosphorylated by bovine cardiac 3':5'-cyclic AMP-dependent protein kinase. Most of the phosphate incorporation was observed in troponin I, the maximum of which was 0.7 mol of Pi per mol of troponin I. 2. In the presence of phosphorylated native tropomyosin, actomyosin ATPase activity was 15-40% lower than that in the presence of the unphosphorylated preparation at all calcium ion concentrations (1.5 x 10(-8) M-2.4 x 10(-5) M). Half-maximum activation of ATPase was obtained with a concentration of 7 x 10(-7) M Ca2+ (unphosphorylated) and 1.3 x 10(-6) M Ca2+ (phosphorylated), respectively. Maximum ATPase activity was reached with 3 x 10(-6) M Ca2+ (unphosphorylated) and 1.0 x 10(-5) M Ca2+ (phosphorylated). 3. Porcine cardiac troponin I isolated by affinity chromatography inhibited ATPase activity of desensitized actomyosin in the presence of tropomyosin. There was little difference between phosphorylated troponin I and a control preparation with regard to the inhibitory effect of ATPase activity. 4. Troponin C from rabbit skeletal muscle neutralized the inhibitory effect of troponin I. The minimum amount of troponin C required for complete neutralization was approximately equimolar to troponin I. The inhibitory effect of phosphorylated troponin I was neutralized by troponin C less effectively than that of unphosphorylated preparation.