Platelet tropomyosin binding protein is identical with serum albumin

We have shown that the platelet tropomyosin binding protein described in the accompanying paper (Gerhard, M. D., DiGirolamo, P. M., and Hitchcock-DeGregori, S. E. (1985) J. Biol. Chem. 260, 3221-3227) is identical with human serum albumin. The immunological determinants are completely shared; the tryptic peptide maps are the same; the proteins comigrate on two-dimensional gels; and the amino acid sequences of the first 33 amino acids are the same. Although human serum albumin in plasma or commercially prepared protein will not bind tropomyosin-Affi-Gel 15, it will bind following purification from plasma by chromatography on hydroxylapatite.     

Effects of the amino-terminal regions of tropomyosin and troponin T on thin filament assembly.

Bacterially expressed alpha-tropomyosin lacks the amino-terminal acetylation present in muscle tropomyosin and binds poorly to actin (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Using a linear lattice model, we determined the affinity (Ko) of unacetylated tropomyosin or troponin-unacetylated tropomyosin for an isolated site on the actin filament and the fold increase in affinity (y) when binding is to an adjacent site. The absence of tropomyosin acetylation decreased Ko 2 orders of magnitude in the absence of troponin. Tropomyosin acetylation also enhanced troponin-tropomyosin binding to actin, not by increasing cooperativity (y), but rather by increasing Ko. These results suggest that the amino-terminal region of tropomyosin is a crucial actin binding site. Troponin promoted unacetylated tropomyosin binding to actin, increasing Ko more than 1,000-fold. Troponin70-259, which lacks the troponin T peptide (1-69) spanning the overlap between adjacent tropomyosins, behaved similarly to intact troponin. Cooperative interactions between adjacent troponin-tropomyosin complexes remained strong despite the use of a nonpolymerizable tropomyosin and a troponin unable to bridge neighboring tropomyosins physically. The Ko for troponin70-259-unacetylated tropomyosin was 500-fold greater than for troponin159-259-unacetylated tropomyosin, indicating that troponin T residues 70-158 are critical for anchoring troponin-tropomyosin to F-actin. The mechanism of cooperative thin filament assembly is discussed.     

The structure of the amino terminus of tropomyosin is critical for binding to actin in the absence and presence of troponin

Analysis of two recombinant variants of chicken striated muscle alpha-tropomyosin has shown that the structure of the amino terminus is crucial for most aspects of tropomyosin function: affinity to actin, promotion of binding to actin by troponin, and regulation of the actomyosin MgATPase. Initial characterization of variants expressed and isolated from Escherichia coli has been published (Hitchcock-DeGregori, S. E., and Heald, R. W. (1987) J. Biol. Chem. 262, 9730-9735). Fusion tropomyosin contains 80 amino acids of a nonstructural influenza virus protein (NS1) on the amino terminus. Nonfusion tropomyosin is a variant because the amino-terminal methionine is not acetylated (unacetylated tropomyosin). The affinity of tropomyosin labeled at Cys190 with N-[14C]ethylmaleimide for actin was measured by cosedimentation in a Beckman Airfuge. Fusion tropomyosin binds to actin with an affinity slightly greater than that of chicken striated muscle alpha-tropomyosin (Kapp = 1-2 X 10(7) versus 0.5-1 X 10(7) M-1) and more strongly than unacetylated tropomyosin (Kapp = 3 X 10(5) M-1). Both variants bind cooperatively to actin. Troponin increases the affinity of unacetylated tropomyosin for actin (+Ca2+, Kapp = 6 X 10(6) M-1; +EGTA, Kapp = 2 X 10(7) M-1), but the affinity is still lower than that of muscle tropomyosin for actin in the presence of troponin (Kapp much greater than 10(8) M-1). Troponin has no effect on the affinity of fusion tropomyosin for actin indicating that binding of troponin T to the over-lap region of the adjacent tropomyosin, presumably sterically prevented by the fusion peptide in fusion tropomyosin, is required for troponin to promote the binding of tropomyosin to actin. The role of troponin T in regulation and the mechanisms of cooperative binding of tropomyosin to actin have been discussed in relation to this work.     

The troponin tail domain promotes a conformational state of the thin filament that suppresses myosin activity

In cardiac and skeletal muscles tropomyosin binds to the actin outer domain in the absence of Ca(2+), and in this position tropomyosin inhibits muscle contraction by interfering sterically with myosin-actin binding. The globular domain of troponin is believed to produce this B-state of the thin filament (Lehman, W., Hatch, V., Korman, V. L., Rosol, M., Thomas, L. T., Maytum, R., Geeves, M. A., Van Eyk, J. E., Tobacman, L. S., and Craig, R. (2000) J. Mol. Biol. 302, 593-606) via troponin I-actin interactions that constrain the tropomyosin. The present study shows that the B-state can be promoted independently by the elongated tail region of troponin (the NH(2) terminus (TnT-(1-153)) of cardiac troponin T). In the absence of the troponin globular domain, TnT-(1-153) markedly inhibited both myosin S1-actin-tropomyosin MgATPase activity and (at low S1 concentrations) myosin S1-ADP binding to the thin filament. Similarly, TnT-(1-153) increased the concentration of heavy meromyosin required to support in vitro sliding of thin filaments. Electron microscopy and three-dimensional reconstruction of thin filaments containing TnT-(1-153) and either cardiac or skeletal muscle tropomyosin showed that tropomyosin was in the B-state in the complete absence of troponin I. All of these results indicate that portions of the troponin tail domain, and not only troponin I, contribute to the positioning of tropomyosin on the actin outer domain, thereby inhibiting muscle contraction in the absence of Ca(2+).     

Chicken cardiac tropomyosin and a low-molecular-weight nonmuscle tropomyosin are related by alternative splicing

We have isolated and characterized complementary DNAs (cDNAs) encoding chicken cardiac muscle tropomyosin and a low-molecular-weight nonmuscle tropomyosin. The cardiac muscle cDNA (pCHT-4) encodes a 284-amino acid protein that differs from chicken skeletal muscle alpha- and beta-tropomyosins throughout its length. The nonmuscle cDNA (pFT-C) encodes a 248-amino acid protein that is most similar (93-94%) to the tropomyosin class including rat fibroblast TM-4, equine platelet tropomyosin, and human fibroblast TM30pl. The nucleotide sequences of the cardiac and nonmuscle cDNAs are identical from the position encoding cardiac amino acid 81 (nonmuscle amino acid 45) through cardiac amino acid 257 (nonmuscle amino acid 221). The sequences differ both 5' and 3' of this region of identity. These comparisons suggest that the chicken cardiac tropomyosin and low-molecular-weight "platelet-like" tropomyosin are derived from the same genomic locus by alternative splicing. S1 analysis suggests that this locus encodes at least one other tropomyosin isoform.     

A 1H NMR study of a ternary peptide complex that mimics the interaction between troponin C and troponin I.

The troponin I peptide N alpha-acetyl TnI (104-115) amide (TnIp) represents the minimum sequence necessary for inhibition of actomyosin ATPase activity of skeletal muscle (Talbot, J.A. & Hodges, R.S. 1981, J. Biol. Chem. 256, 2798-3802; Van Eyk, J.E. & Hodges, R.S., 1988, J. Biol. Chem. 263, 1726-1732; Van Eyk, J.E., Kay, C.M., & Hodges, R.S., 1991, Biochemistry 30, 9974-9981). In this study, we have used 1H NMR spectroscopy to compare the binding of this inhibitory TnI peptide to a synthetic peptide heterodimer representing site III and site IV of the C-terminal domain of troponin C (TnC) and to calcium-saturated skeletal TnC. The residues whose 1H NMR chemical shifts are perturbed upon TnIp binding are the same in both the site III/site IV heterodimer and TnC. These residues include F102, I104, F112, I113, I121, I149, D150, F151, and F154, which are all found in the C-terminal domain hydrophobic pocket and antiparallel beta-sheet region of the synthetic site III/site IV heterodimer and of TnC. Further, the affinity of TnIp binding to the heterodimer (Kd = 192 +/- 37 microM) was found to be similar to TnIp binding to TnC (48 +/- 18 microM [Campbell, A.P., Cachia, P.J., & Sykes, B.D., 1991, Biochem. Cell Biol. 69, 674-681]). The results indicate that binding of the inhibitory region of TnI is primarily to the C-terminal domain of TnC. The results also indicate how well the synthetic peptide heterodimer mimics the C-terminal domain of TnC in structure and functional interactions.     

Characterization of 83-kilodalton nonmuscle caldesmon from cultured rat cells: stimulation of actin binding of nonmuscle tropomyosin and periodic localization along microfilaments like tropomyosin

Nonmuscle caldesmon purified from cultured rat cells shows a molecular weight of 83,000 on SDS gels, Stokes radius of 60.5 A, and sedimentation coefficient (S20,w) of 3.5 in the presence of reducing agents. These values give a native molecular weight of 87,000 and a frictional ratio of 2.04, suggesting that the molecule is a monomeric, asymmetric protein. In the absence of reducing agents, the protein is self-associated, through disulfide bonds, into oligomers with a molecular weight of 230,000 on SDS gels. These S-S oligomers appear to be responsible for the actin-bundling activity of nonmuscle caldesmon in the absence of reducing agents. Actin binding is saturated at a molar ratio of one 83-kD protein to six actins with an apparent binding constant of 5 X 10(6) M-1. Because of 83-kD nonmuscle caldesmon and tropomyosin are colocalized in stress fibers of cultured cells, we have examined effects of 83-kD protein on the actin binding of cultured cell tropomyosin. Of five isoforms of cultured rat cell tropomyosin, tropomyosin isoforms with high molecular weight values (40,000 and 36,500) show higher affinity to actin than do tropomyosin isoforms with low molecular weight values (32,400 and 32,000) (Matsumura, F., and S. Yamashiro-Matsumura. 1986. J. Biol. Chem. 260:13851-13859). At physiological concentration of KCl (100 mM), 83-kD nonmuscle caldesmon stimulates binding of low molecular weight tropomyosins to actin and increases the apparent binding constant (Ka from 4.4 X 10(5) to 1.5 X 10(6) M-1. In contrast, 83-kD protein has slight stimulation of actin binding of high molecular weight tropomyosins because high molecular weight tropomyosins bind to actin strongly in this condition. As the binding of 83-kD protein to actin is regulated by calcium/calmodulin, 83-kD protein regulates the binding of low molecular weight tropomyosins to actin in a calcium/calmodulin-dependent way. Using monoclonal antibodies to visualize nonmuscle caldesmon along microfilaments or actin filaments reconstituted with purified 83-kD protein, we demonstrate that 83-kD nonmuscle caldesmon is localized periodically along microfilaments or actin filaments with similar periodicity (36 +/- 4 nm) as tropomyosin. These results suggest that 83-kD protein plays an important role in the organization of microfilaments, as well as the control of the motility, through the regulation of the binding of tropomyosin to actin.     

Modulation of actin-bundling activity of 55-kDa protein by multiple isoforms of tropomyosin

Cultured rat cells contain five isoforms of tropomyosin (Matsumura, F., Yamashiro-Matsumura, S., and Lin, J.J.-C. (1983) J. Biol. Chem. 258, 6636-6644). To explore the roles of the multiple tropomyosin isoforms in the microfilament organization of cultured cells, we have examined effects of tropomyosins on the bundling activity of the 55-kDa protein recently purified from HeLa cells (Yamashiro-Matsumura, S., and Matsumura, F. (1985) J. Biol. Chem. 260, 5087-5097). Maximum bundling of F-actin was observed at a molar ratio of 55-kDa protein to actin higher than 1:8. None of the isoforms of cultured rat cell tropomyosin significantly altered the F-actin-bundling activity of 55-kDa protein at this ratio, whereas skeletal muscle tropomyosin inhibited the bundling activity to about 50%. Also, cultured cell tropomyosins did not inhibit binding of 55-kDa protein to actin, whereas skeletal muscle tropomyosin inhibited it by 50%. The effect of 55-kDa protein on the binding of tropomyosin to actin varied with the isoform type of tropomyosin. Most (80%) of the tropomyosins with low Mr values (Mr 32,400 or 32,000) were caused to dissociate from actin by 55-kDa protein, but only 20% of tropomyosins with high Mr values (Mr 40,000 or 36,500) was dissociated from actin in these conditions. Immunofluorescence has shown that, while tropomyosin was localized in stress fibers, 55-kDa protein was found in microspikes as well as stress fibers, both of which are known to contain bundles of microfilaments. Therefore, we suggest that 55-kDa protein together with the multiple tropomyosin isoforms may regulate the formation of two types of actin-filament bundles, bundles containing tropomyosin and those without tropomyosin.     

Cooperative interactions between troponin molecules bound to the cardiac thin filament

Striated muscle thin filaments contain many troponin molecules, which contact each other indirectly via tropomyosin and actin. Such allosteric interactions between troponin molecules may be responsible for cooperative Ca2+ binding to the regulatory sites of the cardiac thin filament (Tobacman, L. S., and Sawyer, D. S. (1990) J. Biol. Chem. 265, 931-939). To test whether thin filament-bound troponin molecules interact, we studied the competitive binding of troponin and troponin T-troponin I (an inhibitory complex lacking the Ca2+ binding subunit troponin C) to actin-tropomyosin. The relative affinities of these two forms of troponin for the thin filament depended upon their relative concentrations. Under conditions where total binding was saturated, each form binds with greater apparent affinity to sites that have similar neighbors. A theoretical model for competitive binding of two ligands to interacting sites on a linear lattice was developed and fit to the data. Surprisingly, energetically unfavorable interactions occurred between adjacent troponin and troponin T-troponin I molecules not only in the presence of Ca2+, but also in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid and/or myosin subfragment 1. Removal of Ca2+ strengthened the affinity of troponin for the thin filament less than 50%. These results suggest that, even in the absence of myosin, long range allosteric interactions occur between troponin molecules. The detailed involvement of tropomyosin and actin in these interactions remains to be established.     

Effect of phosphorylation on the interaction and functional properties of rabbit striated muscle alpha alpha-tropomyosin

Phosphorylated rabbit cardiac alpha alpha-tropomyosin has been prepared either enzymatically (Montgomery, K., and Mak, A.S. (1984) J. Biol. Chem. 259, 5555-5560) or by fractionation of the phosphorylated and nonphosphorylated forms on a Mono Q column in 9 M urea, 50 mM Tris, pH 8.0. Although the phosphorylated and nonphosphorylated forms showed no difference in their F-actin binding properties, the phosphorylated protein had substantially higher viscosities at low ionic strengths, indicating a greater propensity for head-to-tail interaction. Similar measurements showed the strengthening of this interaction by whole troponin to be substantially reduced by phosphorylation even though the binding of whole troponin and troponin T to tropomyosin was demonstrated by affinity chromatography to be, if anything, strengthened by phosphorylation. In a reconstituted actin (4 microM) plus myosin subfragment 1 ATPase assay (50 mM ionic strength), significantly higher activities over a range (1 to 8 microM) of subfragment 1 concentrations were observed with phosphorylated tropomyosin compared with the nonphosphorylated protein. In the fully reconstituted system with troponin, there was no significant difference in the inhibition of ATPase in the absence of Ca2+. However, in its presence, the activities were appreciably increased with the phosphorylated tropomyosin compared to those with the nonphosphorylated form. These differences were eliminated by treatment of the phosphorylated tropomyosin with alkaline phosphatase. This is the first demonstration of an effect of phosphorylation on the functional properties of tropomyosin.     

Pig platelet tropomyosin: interactions with the other thin-filament proteins

Pig platelet tropomyosin exhibits many of the functional activities of skeletal tropomyosin. At low ionic strength it forms end-to-end aggregates similar to those formed by skeletal tropomyosins. It forms a 1:1 complex with muscle troponin or with a troponin I-pig brain calmodulin complex, as well as a 1:6 association with platelet filamentous actin. Electron microscopy of paracrystals shows that the troponin binding site is slightly C-terminal of the unique cysteine, corresponding to position 190 of the rabbit skeletal alpha-tropomyosin sequence. The effect of a complex comprising platelet actin and tropomyosin on the ATPase activity of rabbit skeletal muscle myosin subfragment-1 was similar to that displayed by its skeletal muscle counterpart. Platelet tropomyosin decreased the activity by roughly half in a calcium-independent manner. Addition of troponin to the actin-tropomyosin in the absence of calcium results in further inhibition and allows the full activity of the complex to be restored by Ca2+. These results differ from those obtained by C?té & Smillie for horse platelet tropomyosin and this may reflect the different isomeric nature of pig platelet tropomyosin. These results suggest that the functional properties of non-muscle tropomyosins may differ when comparisons are made between proteins isolated from the same type of cell but in different species. Differences in self-association and actin-binding properties may be finely graded between different isoforms.     

A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle

The regulation of striated muscle contraction involves changes in the interactions of troponin and tropomyosin with actin thin filaments. In resting muscle, myosin-binding sites on actin are thought to be blocked by the coiled-coil protein tropomyosin. During muscle activation, Ca2+ binding to troponin alters the tropomyosin position on actin, resulting in cyclic actin-myosin interactions that accompany muscle contraction. Evidence for this steric regulation by troponin-tropomyosin comes from X-ray data [Haselgrove, J.C., 1972. X-ray evidence for a conformational change in the actin-containing filaments of verterbrate striated muscle. Cold Spring Habor Symp. Quant. Biol. 37, 341-352; Huxley, H.E., 1972. Structural changes in actin and myosin-containing filaments during contraction. Cold Spring Habor Symp. Quant. Biol. 37, 361-376; Parry, D.A., Squire, J.M., 1973. Structural role of tropomyosin in muscle regulation: analysis of the X-ray diffraction patterns from relaxed and contracting muscles. J. Mol. Biol. 75, 33-55] and electron microscope (EM) data [Spudich, J.A., Huxley, H.E., Finch, J., 1972. Regulation of skeletal muscle contraction. II. Structural studies of the interaction of the tropomyosin-troponin complex with actin. J. Mol. Biol. 72, 619-632; O'Brien, E.J., Gillis, J.M., Couch, J., 1975. Symmetry and molecular arrangement in paracrystals of reconstituted muscle thin filaments. J. Mol. Biol. 99, 461-475; Lehman, W., Craig, R., Vibert, P., 1994. Ca2+-induced tropomyosin movement in Limulus thin filaments revealed by three-dimensional reconstruction. Nature 368, 65-67] each with its own particular strengths and limitations. Here we bring together some of the latest information from EM analysis of single thin filaments from Pirani et al. [Pirani, A., Xu, C., Hatch, V., Craig, R., Tobacman, L.S., Lehman, W. (2005). Single particle analysis of relaxed and activated muscle thin filaments. J. Mol. Biol. 346, 761-772], with synchrotron X-ray data from non-overlapped muscle fibres to refine the models of the striated muscle thin filament. This was done by incorporating current atomic-resolution structures of actin, tropomyosin, troponin and myosin subfragment-1. Fitting these atomic coordinates to EM reconstructions, we present atomic models of the thin filament that are entirely consistent with a steric regulatory mechanism. Furthermore, fitting the atomic models against diffraction data from skinned muscle fibres, stretched to non-overlap to preclude crossbridge binding, produced very similar results, including a large Ca2+-induced shift in tropomyosin azimuthal location but little change in the actin structure or apparent alteration in troponin position.     

Annealing of gelsolin-severed actin fragments by tropomyosin in the presence of Ca2+. Potentiation of the annealing process by caldesmon

Previous results from our laboratory have shown that 1) cultured rat cells contain two classes of tropomyosin (TM), one (high Mr TMs) with higher Mr values and greater affinity for actin than the other (low Mr TMs); 2) presaturation of F-actin with high Mr TMs, but not with low Mr TMs, inhibits both actin-severing and actin binding activities of gelsolin; and 3) nonmuscle caldesmon not only enhances the inhibitory effects of high Mr TMs but also makes low Mr TMs capable of inhibiting the severing activity of gelsolin (Ishikawa, R., Yamashiro, S., and Matsumura, F. (1989) J. Biol. Chem. 264, 7490-7497). These results suggest that gelsolin has much lower affinity for F-actin-TM-caldesmon complexes than for pure F-actin. We have therefore examined whether addition of TM and/or caldesmon to gelsolin-severed actin filaments can make gelsolin dissociate from barbed ends of actin filaments, resulting in annealing of short actin filaments into long ones. Flow birefringence and electron microscopic studies have suggested that high Mr TMs slowly and partially anneal gelsolin-severed actin fragments in 3 h, whereas low Mr TMs have no effects. Nonmuscle caldesmon greatly potentiates the effects of high Mr TMs and accelerates the process to 20 min, whereas nonmuscle caldesmon alone shows no effects. Furthermore, nonmuscle caldesmon makes low Mr TMs capable of reversing gelsolin-severing action. Actin binding assay has shown that gelsolin (or a gelsolin-actin complex) is dissociated from these annealed actin filaments. Smooth muscle TM and smooth muscle caldesmon also appear to anneal gelsolin-severed actin fragments as do high Mr TMs and nonmuscle caldesmon. Calmodulin decreases the potentiation effects of caldesmon as calmodulin inhibits actin binding of caldesmon. These results suggest that tropomyosin and caldesmon may regulate both capping and severing activities of gelsolin.     

Identification and purification of a novel Mr 43,000 tropomyosin-binding protein from human erythrocyte membranes

A new Mr 43,000 tropomyosin-binding protein (TMBP) has been identified in erythrocyte membranes by binding of 125I-labeled Bolton-Hunter tropomyosin to nitrocellulose blots of membrane proteins separated by sodium dodecyl sulfate-gel electrophoresis. This protein is not actin, because 125I-tropomyosin does not bind to purified actin on blots. Binding of 125I-tropomyosin to this protein is specific because it is inhibited by excess unlabeled tropomyosin but not by F-actin or muscle troponins. This protein has been purified to 95% homogeneity from a 1 M Tris extract of tropomyosin-depleted erythrocyte membranes by DEAE-cellulose and hydroxylapatite chromatography, followed by gel filtration on Ultrogel AcA 44. The purified protein has a Stokes radius of 3.9 nm and a sedimentation coefficient of 2.8 S, corresponding to a native molecular weight of 43,000. Binding of 125I-tropomyosin to the purified TMBP saturates at one tropomyosin molecule (Mr 60,000) to two Mr 43,000 TMBPs, with an affinity of about 5 X 10(-7) M. The TMBP is associated with the membrane skeleton after extraction of membranes with the non-ionic detergent, Triton X-100, and is present with respect to tropomyosin at a ratio of about one for every two tropomyosin molecules. Because there is enough tropomyosin for two tropomyosin molecules to be associated with each of the short actin filaments in the membrane skeleton, the erythrocyte membrane TMBP, together with tropomyosin, could function to restrict the number of spectrin molecules attached to each of the short actin filaments and thus specify the hexagonal symmetry of the spectrin-actin lattice. Alternatively, this TMBP could be homologous to one of the muscle troponins and might function with tropomyosin to regulate erythrocyte actomyosin-ATPase activity and influence erythrocyte shape.     

Tropomodulin isolated from rabbit skeletal muscle inhibits filament formation of actin in the presence of tropomyosin and troponin.

Tropomodulin is a tropomyosin-binding protein, originally isolated from human erythrocytes. Tropomodulin is currently regarded as the sole actin pointed-end capping protein [Weber, A., Pennise, C.R., Babcock, G.G. & Fowler, V.M. (1994) J. Cell Biol. 127, 1627-1635]. This work first describes a procedure for the purification of tropomodulin from rabbit skeletal muscle. Tropomodulin almost completely inhibited filament formation of actin in the presence of tropomyosin and troponin. For the maximal inhibition of actin polymerization, approximately 0.10, 0.12 and 0.003 mol of tropomyosin, troponin and tropomodulin per mol of actin were required, respectively. Fluorescence-intensity measurements, electron-microscopy and sedimentation experiments revealed that only very short fragments and amorphous aggregates, but not filaments, were formed when actin was copolymerized with tropomyosin, troponin and tropomodulin by the addition of 50 mM KCl at pH 8.0. The effects of tropomyosin, troponin and tropomodulin were more remarkable on Ca-actin than on Mg-actin. It appears that tropomodulin caps both the pointed and barbed ends of tropomyosin- and troponin-bound actin filaments.     

Function of the N-terminal calcium-binding sites in cardiac/slow troponin C assessed in fast skeletal muscle fibers

Fast skeletal troponin C (sTnC) has two low affinity Ca(2+)-binding sites (sites I and II), whereas in cardiac troponin C (cTnC) site I is inactive. By modifying the Ca2+ binding properties of sites I and II in cTnC it was demonstrated that binding of Ca2+ to an activated site I alone is not sufficient for triggering contraction in slow skeletal muscle fibers (Sweeney, H.L., Brito, R. M.M., Rosevear, P.R., and Putkey, J.A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 9538-9542). However, a similar study using sTnC showed that Ca2+ binding to site I alone could partially activate force production in fast skeletal muscle fibers (Sheng, Z., Strauss, W.L., Francois, J.M., and Potter, J.D. (1990) J. Biol. Chem. 265, 21554-21560). The purpose of the current study was to examine the functional characteristics of modified cTnC derivatives in fast skeletal muscle fibers to assess whether or not either low affinity site can mediate force production when coupled to fast skeletal isoforms of troponin (Tn) I and TnT. Normal cTnC and sTnC were compared with engineered derivatives of cTnC having either both sites I and II active, or only site I active. In contrast to what is seen in slow muscle, binding of Ca2+ to site I alone recovered about 15-20% of the normal calcium-activated force and ATPase activity in skinned fast skeletal muscle fibers and myofibrils, respectively. This is most likely due to structural differences between TnI and/or TnT isoforms that allow for partial recognition and translation of the signal represented by binding Ca2+ to site I of TnC when associated with fast skeletal but not slow skeletal muscle.     

The N-acetylglucosamine-specific receptor of the thyroid. Binding characteristics, partial characterization, and potential role

The binding characteristics of the GlcNAc binding protein present in thyroid membranes (Consiglio, E., Shifrin, S., Yavin, Z., Ambesi-Impiombato, F.S., Rall, J.E., Salvatore, G., and Kohn, L.D. (1981) J. Biol. Chem. 256, 10592-10599) were reinvestigated using neoglycoproteins as probes. Plasma membrane preparations from porcine thyroid specifically bound 125I-GlcNAc35-bovine serum albumin. Binding was dependent on the presence of calcium. Binding of ligand to receptor was minimal at neutral pH and maximal at pH 5.0. Equilibrium binding studies indicated positive cooperativity of binding and a site capacity of about 60 pmol/mg of protein. Competition studies were compatible with a specificity hierarchy of GlcNAc much greater than Gal; no recognition of mannose, fucose, or glucose moieties was noted. The receptor was detergent-solubilized from plasma membrane preparations and on the basis of the defined binding properties, purified by chromatography on a GlcNAc-Sepharose affinity column. The purified GlcNAc thyroid receptor has a subunit molecular size of about 45 kDa and appears to be an oligomer composed of three subunits. The receptor was identified as a component of thyrocytes by in situ cytochemical localization with fluorescent neoglycoproteins. In certain cases it was mainly present on, or near, the apical cell surface. It is suggested that this GlcNAc receptor functions in thyroglobulin metabolism, possibly involved in recycling of internalized thyroglobulin molecules back into the follicular lumen.     

A proton nuclear magnetic resonance and molecular modeling study of cardiac troponin C. Calcium dependence and aromatic spectral assignments

Proton (1H) NMR at 360 MHz has been used to characterize calcium-induced spectral changes in bovine cardiac troponin C in more detail than hitherto reported (Hincke, M. T., Sykes, B. D., and Kay, C. M. (1981) Biochemistry 20, 3286-3294). The observed changes are consistent with two equivalents of calcium occupying high affinity sites, with subsequent binding of a single equivalent to a lower affinity site. Two-dimensional J-correlated and nuclear Overhauser effect NOE-correlated and conventional one-dimensional NOE experiments, combined with a consideration of the titration behavior, have allowed all the aromatic signals, and several prominently shifted alpha-CH and methyl group signals, as well as some methionine methyl signals of the calcium-saturated protein, to be assigned. This exercise was facilitated by the construction of a model of the calcium-bound protein based on crystal structure data of the homologous calmodulin and skeletal troponin C, using mutations, energy minimizations, and molecular dynamics simulations, combined with the ring-current shift and NOE prediction program PARSNIP (Reid, D. G., and Saunders, M. R. (1989) J. Biol. Chem. 264, 2003-2012).     

Ca-binding and Ca-sensitizing functions of cardiac native tropomyosin, troponin, and tropomyosin

Procedures are described by which troponin and tropomyosin can be isolated from cardiac muscle rapidly, with minimal damage by oxidation. Cardiac relaxing proteins inhibit actomyosin ATPase activity in the presence of ethyleneglycoltetraacetic acid (EGTA), and permit graded stimulation by Ca²⁺. This stimulation is independent of preexisting inhibition, and greater than that obtained with skeletal proteins. Characteristics of Scatchard plots for Ca²⁺ binding suggest that troponin contains one class of sites which interact at high fractional occupancy. Interaction appears to be enhanced by tropomyosin. Mean values for the estimated maximum affinity and capacity of six canine cardiac troponin preparations were: 4.92·10⁶ M⁻¹, and 21.58·10⁻⁶ moles·g⁻¹. Values for skeletal troponin were not significantly different. Native tropomyosin bound about half as much Ca²⁺ per g, with maximum affinity the same as troponin. Pure tropomyosin bound no Ca²⁺. Cardiac and skeletal proteins differ in that the former are much more labile, and more readily influenced by ions and drugs.     

Fluorescence resonance energy transfer within the complex formed by actin and myosin subfragment 1. Comparison between weakly and strongly attached states

Fluorescence resonance energy transfer measurements have been made between Cys-374 on actin and Cys-177 on the alkali light chain of myosin subfragment 1 (S1) using several pairs of donor-acceptor chromophores. The labeled light chain was exchanged into subfragment 1 and the resulting fluorescently labeled subfragment 1 isolated by ion-exchange chromatography on SP-Trisacryl. The efficiency of energy transfer was measured by steady-state fluorescence in a strong binding complex of acto-S1 and found to represent a spatial separation between the two probes of 5.6-6.3 nm. The same measurements were then made with weak binding acto-S1 complexes generated in two ways. First, actin was complexed with p-phenylenedimaleimide-S1, a stable analogue of S1-adenosine 5'-triphosphate (ATP), obtained by cross-linking the SH1 and SH2 heavy-chain thiols of subfragment 1 [Greene, L. E., Chalovich, J. M., & Eisenberg, E. (1986) Biochemistry 25, 704-709]. Large increases in transfer efficiency indicated that the two probes had moved closer together by some 3 nm. Second, weak binding complexes were formed between subfragment 1 and actin in the presence of the regulatory proteins troponin and tropomyosin, the absence of calcium, and the presence of ATP [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437]. The measured efficiency of energy transfer again indicated that the distance between the two labeled sites had moved closer by about 3 nm. These data support the idea that there is a considerable difference in the structure of the acto-S1 complex between the weakly and strongly bound states.