Skeletal Troponin C Research Articles

Evidence that both Ca(2+)-specific sites of skeletal muscle TnC are required for full activity.

To investigate the role of the Ca2(+)-specific (I and II) sites of fast skeletal muscle troponin C (TnC) in the regulation of contraction, we have produced two TnC mutants which have lost the ability to bind Ca2+ at either site I (VG1) or at site II (VG2). Both mutants were able to partially restore force to TnC-depleted skinned muscle fibers (approximately 25% for VG1 and approximately 50% for VG2). In contrast, bovine cardiac TnC (BCTnC), which like VG1 binds Ca2+ only at site II, could fully reactivate the contraction of TnC-depleted fibers. Higher concentrations of both mutants were required to restore force to the TnC-depleted fibers than with wild type TnC (WTnC) or BCTnC. VG1 and VG2 substituted fibers could not bind additional WTnC, indicating that all of the TnC-binding sites were saturated with the mutant TnC's. The Ca2+ concentration required for force activation was much higher for VG1 and VG2 substituted fibers than for WTnC or BCTnC substituted fibers. Also, the steepness of force activation was much less in VG1 and VG2 versus WTnC and BCTnC substituted fibers. These results suggest cooperative interactions between sites I and II in WTnC. In contrast, BCTnC has essentially the same apparent Ca2+ affinity and steepness of force activation as does WTnC. Thus, cardiac TnC must have structural differences from WTnC which compensate for the lack of site I, while in WTnC, both Ca2(+)-specific sites are probably crucial for full functional activity.

Cloning, structural analysis, and expression of the human fast twitch skeletal muscle troponin C gene.

The gene encoding human fast skeletal muscle troponin C (TnC) was cloned, mapped, and sequenced. The locations of intron positions in this gene were compared to those in the related genes for mouse slow skeletal TnC and vertebrate and nonvertebrate calmodulins. We detected strikingly similar purine-rich DNA sequences on the coding strand in the basal promoter of the genes for fast and slow troponin C and chicken calmodulin II which may represent conserved regulatory elements in genes of the vertebrate troponin C/calmodulin gene family. We mapped the transcriptional start site of the gene and analyzed the expression of TnC test genes in the myogenic cell lines C2, L8, and H9c2(2-1) and in the human fibroblast line HuT12. Constructs comprising 4.7 or 6.2 kilobase pairs of 5'-flanking sequence (including the genuine transcriptional start site) upstream of the chloramphenicol acetyltransferase gene as reporter expressed the hybrid gene in C2 cells but not in nonmuscle cells. Surprisingly, no expression was found in cell lines L8 and H9c2(2-1) despite the fact that all three muscle cell lines vigorously express the endogenous TnC fast mRNA after differentiation. The discrepancy between the expression of endogenous genes and the test gene in these cell lines indicates different requirements for regulatory elements in different myogenic cells.

Ca2(+)-dependent interactions between the C-helix of troponin-C and troponin-I. Photocross-linking and fluorescence studies using a recombinant troponin-C.

We have used in vitro mutagenesis to synthesize in Escherichia coli a recombinant rabbit skeletal troponin-C (designated as TnC57) in which Cys-98 was replaced with leucine, and Ala-57 in the C-helix of the N-terminal domain was replaced with cysteine. TnC57 labeled with the bifunctional photocross-linker benzophenone-4-maleimide could be photocross-linked with troponin-I in both the binary complex with troponin-I and in the ternary complex with troponin-I and troponin-T. The fluorescence lifetime of TnC57 labeled with the probe N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine decreased from 13.2 +/- 0.1 to 11.8 +/- 0.1 ns when Ca2+ bound to the low affinity triggering sites. Complexation with either troponin-I or both troponin-I and troponin-T resulted in significant increases in this lifetime both in the absence and the presence of Ca2+. In either the binary or the ternary complex, this lifetime increased from 15.5 to 18.0 ns upon Ca2+ binding to the low affinity sites. Complementary acrylamide-quenching studies yielded results that are consistent with the fluorescence lifetime results. Our results show that the C-helix of troponin-C interacts with troponin-I, in confirmation of recent zero-length cross-linking results (Leszyk, J., Grabarek, Z., Gergely, J., and Collins, J.H. (1990) Biochemistry 29, 299-304). Moreover, they are in support of a model (Herzberg, O., Moult, J., and James, M.N.G. (1986) J. Biol. Chem. 261, 2638-2644) in which the binding of Ca2+ to the triggering sites in the N-terminal domain of troponin-C results in the movement of the B- and C-helices away from the central helix, thereby exposing a putative hydrophobic binding site for troponin-I.

Modification of temperature dependence of myofilament Ca sensitivity by troponin C replacement

The Ca sensitivity of chemically skinned right ventricular trabeculae from the rat heart was determined at 22 and 8 degrees C. Endogenous troponin C (TnC) was then extracted with EDTA and replaced with either bovine cardiac TnC or rabbit fast-twitch skeletal TnC. The temperature dependence of myofilament Ca sensitivity was then reevaluated. Cooling native cardiac tissue from 22 to 8 degrees C reduced the pCa (-log10 [Ca2+]), generating half-maximal tension (K1/2) from 5.20 +/- 0.07 to 4.89 +/- 0.08 (SD, n = 14), and also reduced maximum Ca-activated force to 33 +/- 6% of its value at 22 degrees C. After extraction of endogenous TnC and reconstitution with cardiac TnC, cooling from 22 to 8 degrees C caused a similar shift in mean K1/2 from 4.93 +/- 0.08 to 4.69 +/- 0.06 (n = 7). When skeletal TnC was reconstituted into TnC-extracted ventricular fibers, cooling from 22 to 8 degrees C led to a much smaller mean shift in K1/2 from 4.88 +/- 0.07 to 4.78 +/- 0.04 (n = 7). The results show that the magnitude of the cooling-induced shift in myofilament Ca sensitivity observed in the native state (or after reconstitution with cardiac TnC) is significantly reduced if the fiber is reconstituted with skeletal TnC (P less than 0.001). This indicates that the temperature dependence of myofilament Ca sensitivity of cardiac muscle can be modified by incorporation of skeletal TnC. Thus Ca binding to TnC plays an important role in determining the temperature dependence of myofilament Ca sensitivity.

Characterization of zero-length cross-links between rabbit skeletal muscle troponin C and troponin I: evidence for direct interaction between the inhibitory region of troponin I and the amino-terminal, regulatory domain of troponin C

Interactions between troponin C (TnC) and troponin I (TnI) play an important role in the Ca2(+)-dependent regulation of vertebrate striated muscle contraction. Previous attempts to elucidate the molecular details of TnC-TnI interactions, mainly involving chemically modified proteins or fragments thereof, have led to the widely accepted idea that the "inhibitory region" (residues 96-116) of TnI binds to an alpha-helical segment of TnC comprising residues 89-100 in the nonregulatory, COOH-terminal domain. In an attempt to identify other possible physiologically important interactions between these proteins, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC) was used to produce zero-length cross-links in the complex of rabbit skeletal muscle TnC and TnI. TnC was activated with EDC and N-hydroxysuccinimide (NHS) and then mixed with an equimolar amount of TnI [Grabarek, Z., & Gergely, J. (1988) Biophys. J. 53, 392a]. The resulting cross-linked TnCXI was cleaved with cyanogen bromide, trypsin, and Staphylococcus aureus V8 protease (SAP). Cross-linked peptides were purified by reverse-phase HPLC and characterized by sequence analysis. The results indicated that residues from the regulatory Ca2(+)-binding site II in the NH2-terminal domain of TnC (residues 46-78) formed cross-links with TnI segments spanning residues 92-167. The most highly cross-linked residues in TnI were Lys-105 and Lys-107, located in the inhibitory region. These results yield the first evidence for an interaction between the N-terminal domain of TnC and the inhibitory region of TnI.

Molecular cloning and expression of chicken cardiac troponin C.

We have isolated a full length complementary DNA clone (pCTnC1) from a 19-day embryonic chicken heart library corresponding to cardiac troponin C (TnC). Sequence analysis demonstrated varying homologies with TnC complementary DNA clones isolated from developing chick skeletal muscle. Using pCTnC1 as a hybridization probe, we have determined that cardiac TnC is constitutively expressed in both atria and ventricles of the developing and adult heart. These data along with previous immunochemical studies of TnC expression demonstrate that the slow skeletal and cardiac muscle isoform is the only TnC expressed in the heart. In contrast, expression of slow skeletal and cardiac muscle TnC is developmentally regulated in skeletal muscles of the chicken. The tightly controlled expression of slow skeletal and cardiac muscle TnC in the varying myocyte types of the heart suggests a physiologically significant role of this regulatory protein.

Ca2+, pH and the regulation of cardiac myofilament force and ATPase activity

When the pH surrounding myofilaments of striated muscle is reduced there is an inhibition of both the actin-myosin reaction as well as the Ca2+-sensitivity of the myofilaments. Although the mechanism for the effect of acidic pH on Ca2+-sensitivity has been controversial, we have evidence for the hypothesis that acidic pH reduces the affinity of troponin C (TNC) for Ca2+. This effect of acidic pH depends not only on a direct effect of protons on Ca2+-binding to TNC, but also upon neighboring thin filament proteins, especially TNI, the inhibitory component of the TN complex. Using fluorescent probes that report Ca2+-binding to the regulatory sites of skeletal and cardiac TNC, we have shown, for example, that acidic pH directly decreases the Ca2+-affinity of TNC, but only by a relatively small amount. However, with TNC in whole TN or in the TNI-TNC complex, there is about a 2-fold enhancement of the effects of acidic pH on Ca2+-binding to TNC. Acidic pH decreases the affinity of skeletal TNI for skeletal TNC, and also influences the micro-environment of a probe positioned at Cys-133 of TNI, a region of interaction with TNC. Other evidence that the effects of acidic pH on Ca2+-TNC activation of myofilaments are influenced by TNI comes from studies with developing hearts. In contrast, to the case with the adult preparations, Ca2+-activation of detergent extracted fibers prepared from dog or rat hearts in the peri-natal period are weakly affected by a drop in pH from 7.0 to 6.5. This difference in the effect of acidic pH appears to be due to a difference in the isoform population of TNI, and not to differences in isotype population or amount of TNC.

Isolation and Characterization of cDNA Clones Encoding Embryonic and Adult Isoforms of Rat Cardiac Troponin T

We have previously reported that troponin T (TnT), a regulatory protein in muscle contraction, undergoes a switch from the larger, acidic embryonic form to the smaller, basic adult form during rat heart development (Jin, J.-P., and Lin, J.J.-C. (1988) J. Biol. Chem. 263, 7309-7315). To investigate the significance and the molecular mechanism of this isoform switching, cDNA clones encoding rat cardiac TnT were obtained by screening a cDNA library constructed from young rat cardiac poly(A)+ RNAs using the expression vector lambda gt11. Clone RCT10 proved to be a full length clone containing 50 base pairs (bp) of 5'-untranslated sequence, 870 bp of coding sequence, and 196 bp plus poly(A) tail at the 3'-untranslated region. In the other cDNA clone (RCT11), the entire 3'-untranslated sequence and most of the coding sequence were identical with that of RCT10, but an additional 30-bp insert was present in the coding region from residues 18 to 27. This insertion sequence appeared to code for a fragment (EDWSEEEEDE) highly enriched in acidic amino acid residues. Thus, RCT11 might represent a clone encoding the embryonic isoform of rat cardiac TnT. S1 nuclease RNA mapping analysis using end-labeled RCT11 cDNA probes confirmed that this region of the sequence is different in embryonic and adult isoform mRNAs. These results suggest that both embryonic and adult isoforms of rat cardiac TnT are generated from the same primary transcript by developmentally regulated alternative splicing. The amino acid sequence deduced from RCT10 cDNA exhibits 87%, 85%, and 72% homology with bovine, rabbit, and chicken cardiac TnTs, respectively, but less homology (57-59%) with the known skeletal TnTs from human, rat, rabbit, and chicken. Moreover, both the 5'- and the 3'-untranslated sequences of rat cardiac TnT mRNA are completely different from those reported for rat skeletal TnT mRNA, suggesting that rat cardiac TnT is coded from a gene distinct from the rat skeletal TnT gene.

Differential expression of slow and fast skeletal muscle troponin C: Slow skeletal muscle troponin C is expressed in human fibroblasts

We have isolated and sequenced the cDNAs for human slow and fast skeletal muscle troponin C (TnC). Each cDNA is encoded by one of the two TnC genes in the human genome. The fast skeletal muscle TnC gene appears to be expressed exclusively in skeletal muscle. Only the slow TnC gene is expressed in human cardiac ventricle. The slow skeletal TnC gene is also expressed in skeletal muscle and, surprisingly, in several human fibroblast cell lines. Thus, at least one of the three proteins of the troponin complex appears to be expressed in non-muscle cells of higher vertebrates. The relative steady-state amounts of the slow and fast skeletal TnC mRNAs in various adult and embryonic striated muscles are similar to the expected amounts of the corresponding protein, suggesting that the expression of TnC genes is controlled predominantly by the production or accumulation of mRNA rather than by translational or post-translational mechanisms.

Skinned ventricular fibres: troponin C extraction is species-dependent and its replacement with skeletal troponin C changes Sr2+ activation properties

Troponin C (TnC) was extracted from bundles of rat and mouse skinned cardiac ventricular cells by a method similar to that previously used to extract TnC from skinned rabbit skeletal muscle cells (Kerrick et al., J. biol. Chem. 260, 15687-93, 1985) and replaced with either bovine cardiac or rabbit fast-twitch skeletal TnC. In contrast, the same TnC extraction conditions will not extract TnC from either bovine or rabbit cardiac skinned fibres. When the extracted TnC was replaced by bovine cardiac TnC the Sr2+-activated tension relationship was not altered from control values. In contrast, replacement of the endogenous TnC with exogenous rabbit fast-twitch TnC caused the relationship between Sr2+ concentration and tension to shift towards higher concentrations of Sr2+. The Sr2+-activated tension of rat fibres with fast-twitch TnC was identical to that of rabbit fast-twitch skinned fibres. Partial skeletal TnC substitution in skinned cardiac cells gradually shifted the relationship between tension and [Sr2+] to higher Sr2+ concentrations and caused the cells to be activated over a wider range of Sr2+ concentrations. Thus it appears that the activation of rat cardiac skinned cells by Sr2+ is determined by characteristics of the TnC. In contrast, the Sr2+ activation of skinned rabbit fast-twitch skeletal fibres containing either cardiac or skeletal TnC is identical, strongly suggesting that protein-protein interactions determined the Sr2+ -activation properties in these fibres.

Troponin I enhances acidic pH-induced depression of Ca2+ binding to the regulatory sites in skeletal troponin C.

Inhibition of muscle force development by acidic pH is a well known phenomenon, yet the exact mechanism by which a decrease in pH inhibits the Ca2+-activated force in striated myofilaments remains poorly understood. Whether or not the deactivation by acidic pH involves direct competition between Ca2+ and protons for regulatory binding sites on fast skeletal troponin C (TnC) or whether other proteins in thin filament regulation are important remains unclear. We measured the effects of acidic pH on Ca2+-dependent fluorescent changes in TnC labeled with the probe danzylaziridine (Danz), which reports Ca2+ binding to the regulatory (Ca2+-specific) sites. Measurements were also made with TnCDanz complexed with the inhibitory Tn unit, TnI, and in the whole Tn complex. Our results show that a drop in pH from 7.0 to 6.5 is associated with a 1.6-fold increase in the midpoint for the relation between free Ca2+ and Ca2+ binding to the regulatory sites on TnCDanz. However, when TnCDanz was present in its complex with either TnI alone or with TnI-TnT, the increase in midpoint free Ca2+ was increased by 3.5-fold. We tested whether this potentiation in the effect of acidic pH on Ca2+ binding to TnC is due to a pH-induced alteration in the binding of TnI to TnC. A decrease in pH from 7.0 to 6.5 was associated with a halving of the affinity of TnI for TnC. We also probed the effect of acidic pH on TnI. This was done (i) by measuring the intrinsic fluorescence of tryptophan residues in TnI alone and (ii) by measuring fluorescence of TnI (in the Tn complex) labeled at Cys-133 with 5-iodoacetamidofluorescein. A drop in pH from 7.0 to 6.5 was associated with a 15% decrease in intrinsic fluorescence and with a 30% decrease in the fluorescence of the 5-iodoacetamidofluorescein probe. We conclude, therefore, that while protons and Ca2+ may directly affect Ca2+ binding to regulatory sites on fast skeletal TnC, the effect of acidic pH on TnC Ca2+ binding is amplified in the TnI-TnC and Tn complexes by a pH-related effect on the affinity of TnI for TnC.

Cloning, sequencing and expression of a full-length rabbit fast skeletal troponin-C cDNA

A full-length cDNA coding for troponin-C isolated from an adult rabbit fast skeletal muscle library has been sequenced and expressed in an E. coli host. The amino acid sequence derived from the coding region agrees with the published protein sequence except that the first two residues are reversed. The expressed protein was found to be identical to purified rabbit skeletal troponin-C based on electrophoretic mobilities in polyacrylamide gels containing either SDS or urea, and on immunoblotting. These results establish that our troponin-C cDNA clone is suitable for site-directed mutagenesis studies on the structure and function of troponin-C

Two trifluoperazine‐binding sites on calmodulin predicted from comparative molecular modeling with troponin‐C

Among the known regulatory proteins that are conformationally sensitive to the binding of calcium ions, calmodulin and troponin-C have the greatest primary sequence homology. This observation has led to the conclusion that the most accurate predicted molecular model of calmodulin would be based on the X-ray crystallographic coordinates of the highly refined structure of turkey skeletal troponin-C. This paper describes the structure of calmodulin built from such a premise. The resulting molecular model was subjected to conjugate gradient energy minimization to remove unacceptable intramolecular non-bonded contacts. In the analysis of the resulting structure, many features of calmodulin, including the detailed conformation of the Ca2+-binding loops, the amino- and carboxy-terminal hydrophobic patches of the Ca2+-bound form, and the several clusters of acidic residues can be reconciled with much of the previously published solution data. Calmodulin is missing the N-terminal helix characteristic of troponin-C. The deletion of three residues from the central helical linker (denoted D/E in troponin-C) shortens the molecule and changes the orientation of the two domains of calmodulin by 60 degrees relative to those in troponin-C. The molecular model has been used to derive two possible binding sites for the antipsychotic drug trifluoperazine, a potent competitive inhibitor of calmodulin activity.

Calmidazolium, a calmodulin antagonist, stimulates calcium-troponin C and calcium-calmodulin-dependent activation of striated muscle myofilaments.

Although regulatory Ca2+-binding domains of calmodulin (CaM) and troponin C (TnC) are similar, it is interesting that agents that act as CaM antagonists appear to be TnC "agonists" in that they sensitize cardiac myofilaments to activation by Ca2+ (El-Saleh, S., and Solaro, R. J. (1987) Biophys. J. 51, 325 (abstr.). This indicates that the effects of agents that react with Ca2+-binding proteins may depend on protein-protein interactions involved in a particular Ca2+-dependent process. In experiments described here, we have explored this idea by testing effects of calmidazolium (CDZ), a potent calmodulin antagonist on striated muscle myofilaments regulated by cardiac TnC, skeletal TnC, and CaM. CDZ was shown to increase submaximal calcium activation of myofilament force and ATPase activity in both cardiac and skeletal muscle, but the effect was greater in the case of the cardiac preparations. In the presence of 10 microM CDZ, the free Ca2+ giving half-maximal activation was reduced to about 60% of the control value in the case of cardiac myofilaments. Analogous differential effects of CDZ were also seen in studies in which we measured direct effects of CDZ on Ca2+-dependent fluorescence changes of cardiac TnC and skeletal TnC labeled with probes reporting Ca2+ binding to the regulatory sites. Measurements were also done with myofibrillar preparations of psoas muscle in which the native skeletal TnC was removed and exchanged with cardiac TnC and CaM, both of which could substitute for skeletal TnC as a regulatory protein. CDZ was more effective in sensitizing Ca2+-dependent MgATPase activity of skeletal myofibrils containing CaM than in preparations containing the native TnC. However, CDZ was most effective in its Ca2+-sensitizing effect in the case of the preparations containing cardiac TnC. Our results indicate that effects of agents that bind to Ca2+-binding proteins depend not only on the particular variant, but also on the specific environment in which the Ca2+-binding proteins operate.

Kinetics of the conformational change of skeletal troponin C induced by Ca2+-Mg2+ exchange at the high-affinity Ca2+-binding sites.

The rate constant of the conformational change of skeletal troponin C (TnC) induced by the Ca2+ binding reaction with the high-affinity Ca2+-binding sites was determined in the presence of Mg2+ by the fluorescence stopped-flow method in 0.1 M KCl, 50 mM Na-cacodylate-HCl pH 7.0 at 20 degrees C. The [MgCl2] dependence of the rate constants of the observed biphasic conformational change leveled off at the high [MgCl2] region: the rate constants were 60 +/- 9 s-1 and 8 +/- 2 s-1, respectively. These values are larger than the rate constants of the biphasic fluorescence intensity change of TnC induced by Mg2+ removal reaction at the high-affinity Ca2+-binding sites (37 +/- 7 s-1 and 3.0 +/- 0.6 s-1) under the same experimental conditions. These results suggest that the Ca2+-Mg2+ exchange reaction at the high-affinity Ca2+-binding sites is faster than the resultant conformational change accompanying the fluorescence intensity change. Based on these results, we also reexamine the molecular kinetic mechanism of the conformational change of the protein induced by the Mg2+ binding or removal reaction with the high affinity Ca2+-binding sites of skeletal TnC.

A comparative molecular model of the Ca2+sensitive regulatory protein calmodulin based on turkey skeletal troponin-C

A comparative molecular model of the Ca²⁺sensitive regulatory protein calmodulin based on turkey skeletal troponin-C

Calcium-binding properties of troponin C in detergent-skinned heart muscle fibers.

In order to obtain information with regard to behavior of the Ca2+ receptor, troponin C (TnC), in intact myofilament lattice of cardiac muscle, we investigated Ca2+-binding properties of canine ventricular muscle fibers skinned with Triton X-100. Analysis of equilibrium Ca2+-binding data of the skinned fibers in ATP-free solutions suggested that there were two distinct classes of binding sites which were saturated over the physiological range of negative logarithm of free calcium concentration (pCa): class I (KCa = 7.4 X 10(7) M-1, KMg = 0.9 X 10(3) M-1) and class II (KCa = 1.2 X 10(6) M-1, KMg = 1.1 X 10(2) M-1). The class I and II were considered equivalent, respectively, to the Ca2+-Mg2+ and Ca2+-specific sites of TnC. The assignments were supported by TnC content of the skinned fibers determined by electrophoresis and 45Ca autoradiograph of electroblotted fiber proteins. Dissociation of rigor complexes by ATP caused a downward shift of the binding curve between pCa 7 and 5, an effect which could be largely accounted for by lowering of KCa of the class II sites. When Ca2+ binding and isometric force were measured simultaneously, it was found that the threshold pCa for activation corresponds to the range of pCa where class II sites started to bind Ca2+ significantly. We concluded that the low affinity site of cardiac TnC plays a key role in Ca2+ regulation of contraction under physiological conditions, just as it does in the regulation of actomyosin ATPase. Study of kinetics of 45Ca washout from skinned fibers and myofibrils revealed that cardiac TnC in myofibrils contains Ca2+-binding sites whose off-rate constant for Ca2+ is significantly lower than the Ca2+ off-rate constant hitherto documented for the divalent ion-binding sites of either cardiac/slow muscle TnC or fast skeletal TnC.

Troponin-T and glyceraldehyde-3-phosphate dehydrogenase share a common antigenic determinant

A 37 kDa protein in extracts of bovine aorta and equine platelets was observed on SDS-polyacrylamide gel electrophoretograms to react with polyclonal and monoclonal antibodies to rabbit skeletal troponin-T (TnT) by immunoblotting. Following purification by precipitation at pH 4.6 and several ion-exchange chromatographic steps, it has been identified as glyceraldehyde-3-phosphate dehydrogenase (G3PD) by amino acid analyses and NH2-terminal sequencing. By ELISA, the anti-troponin-T monoclonal antibody reacted with rabbit skeletal G3PD appreciably but 120-fold less specifically than with TnT. A cyanogen bromide fragment (CB2) of TnT (residues 71-151) reacted with the monoclonal antibody nearly as well as intact TnT. This cross-reactivity between G3PD and TnT can be ascribed to a weak homology in the amino acid sequences of the two proteins between residues 72-80 of TnT and residues 157-165 of G3PD. Other regions of limited sequence similarity in the two proteins are also present. We conclude that the identification of diffuse cytoplasmic indirect immunofluorescent staining observed with a monoclonal anti-TnT antibody in chicken gizzard muscle is probably attributable to cross-reactivity with G3PD.

Sequential volume changes on a small sample by density

A procedure is described for generating a sequence of volume changes by density on the same sample of a solution. The entire volume-change profile over the reactant concentration range of interest can be accomplished with 0.1–0.2 μmol of a protein in 100 μl of sample. The calcium-binding proteins, calmodulin and skeletal troponin-C, were employed to determine the volume increases attending the sequential addition of calcium ions. Both volume-change profiles exhibited nonlinear increases over the first four equivalents of calcium ion added.

Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C.

Binding of Ca2+ to the troponin C (TnC) subunit of troponin is necessary for tension development in skeletal and cardiac muscles. Tension was measured in skinned fibers from rabbit skeletal muscle at various [Ca2+] before and after partial substitution of skeletal TnC with cardiac TnC. Following substitution, the tension-pCa relationship was altered in a manner consistent with the differences in the number of low-affinity Ca2+-binding sites on the two types of TnC and their affinities for Ca2+. The alterations in the tension-pCa relationship were for the most part reversed by reextraction of cardiac TnC and readdition of skeletal TnC into the fiber segments. These findings indicate that the type of TnC present plays an important role in determining the Ca2+ dependence of tension development in striated muscle.