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The increase in fluorescence of dansylaziridine (DANZ) labeled troponin C (TnCDANZ) substituted into skinned rabbit psoas fibers was determined as a function of the pCa. The fluorescence data are expressed as the ratio of two wavelength bands, one that sees the fluorescence of TnCDANZ, and one that sees background fluorescence and scatter. The percent TnC replaced with TnCDANZ was varied between 10 and 50% and, the fibers were randomly stretched, at the start of each experiment, between 10 and 50%. A large ratio increase accompanies increase in [Ca2+]. The pCa/force data are best fit by the Hill equation but the pCa/ratio data are best fit by a model in which Ca2+ binds in two phases. The position of the force curve on the pCa axis varies little between fibers, in contrast to that of the ratio or Δ-fluorescence curve. In accord with previous reports the Δ-fluorescence can be left of the force on the pCa axis (type I) or superimpose in part on the force (type II). Not described previously, we find curves in which the second phase of the ratio cross-over the pCa/force curve. This type III relationship is found only in fibers less than 3 weeks postmuscle harvest. We propose that the first, relatively invariant, phase of the biphasic pCa/ratio curve accompanies Ca2+ binding to either of the two low affinity sites on TnCDANZ as it does for TnC in solution. The second, highly cooperative, phase of the ratio curve that accompanies muscle contraction and enhanced Ca2+ binding is initiated when sufficient Ca2+ is bound to overcome inhibitory systems. Loose coupling between the initial Ca2+ binding and the cooperative switch point may account for much of the variation in the shape and position of the pCa/ratio curve. There is evidence that, in the overlap zone, weakly attached myosin cross-bridges enhance cooperation between the regulatory units of the thin filaments.

Extraction of troponin C (TnC) from skinned muscle fibers reduces maximum Ca2+ and rigor cross-bridge (RXB)-activated tensions and reduces cooperativity between neighboring regulatory units (one troponin-tropomyosin complex and the seven associated actins) of thin filaments. This suggests that TnC has a determining role in RXB, as well as in Ca(2+)-dependent activation processes. To investigate this possibility further, we replaced fast TnC (fTnC) of rabbit psoas fibers with either CaM[3,4TnC] or cardiac TnC (cTnC) and compared the effects of these substitutions on Ca2+ and RXB activation of tension. CaM[3,4TnC] substitution has the same effect on Ca(2+)- and RXB-activated tensions; they are reduced 50%, and cooperativity between regulatory units is reduced 40%. cTnC substitution also reduces the maximum Ca(2+)-activated tension and cooperativity. But with RXB activation the effects on tension and cooperativity are opposite; cTnC substitution potentiates tension but reduces cooperativity. We considered whether tension potentiation could be explained by increased activation by cycling cross-bridges (CXBs), but the concerted transition formalism predicts fibers will fail to relax in high substrate and high pCa when CXBs are activator ligands. It predicts resting tension, which is not observed in either control or cTnC-substituted fibers. Rather, it appears that cTnC facilitates RXB activation of fast fibers more effectively than fTnC. The order of RXB-activated tension facilitation is cTnC > fTnC > CaM[3,4TnC] > empty TnC-binding sites. Comparison of the structures of fTnC, CaM[3,4TnC], and cTnC indicates that the critical region for this property lies in the central helix or N-terminal domain, including EF hand motifs 1 and 2.

Hodges and his colleagues identified a 12 amino acid fragment of troponin I (TnI-ip) that inhibits Ca(2+)-activated force and reduces the effectiveness Ca(2+) as an activator. To understand the role of troponin C (TnC) in the extended cooperative interactions of thin filament activation, we compared the effect of TnI-ip with that of partial troponin TnC extraction. Both methods reduce maximal Ca(2+)-activated force and increase [Ca(2+)] required for activation. In contrast to TnC extraction, TnI-ip does not reduce the extended cooperative interactions between adjacent thin filament regulatory units as assessed by the slope of the pCa/force relationship. Additional evidence that TnI-ip does not interfere with extended cooperativity comes from studies that activate muscle by rigor crossbridges (RXBs). TnI-ip increases both the cooperativity of activation and the concentration of RXBs needed for maximal force. This shows that TnI-ip binding to TnC increases the stability of the relaxed state of the thin filament. TnI-ip, therefore, uncouples force generation from extended cooperativity in both Ca(2+) and RXB activated muscle contraction. Because maximum force can be reduced with no change-or even an increase-in cooperativity, force-generating crossbridges do not appear to be the primary activators of cooperativity between thin filament regulatory units of skeletal muscle.

SUMMARYAlthough muscle specialization has been studied extensively in vertebrates,less is known about the mechanisms that have evolved in invertebrate muscle that modulate muscle performance. Recent research on the musculature of squid suggests that the mechanisms of muscle specialization in cephalopods may differ from those documented in vertebrates. Muscle diversity in the development and the evolution of cephalopods appears to be characterized by modulation of the dimensions of the myofilaments, in contrast to the relatively fixed myofilament dimensions of vertebrate muscle. In addition, the arrangement of the myofilaments may also be altered, as has been observed in the extensor muscle fibres of the prey capture tentacles of squid and cuttlefish, which show cross-striation and thus differ from the obliquely striated pattern of most cephalopod locomotor muscle fibres. Although biochemical specializations that reflect differences in aerobic capacity have been documented previously for specific layers of the mantle muscle of squid,comparison of protein profiles of myofilament preparations from the fast cross-striated tentacle fibres and slow obliquely striated fibres from the arms has revealed remarkably few differences in myofilament lattice proteins. In particular, previous studies using a variety of SDS-PAGE techniques and peptide mapping of the myosin heavy chain were unable to resolve differences in the myosin light and heavy chains. Since these techniques cannot exclude the presence of a highly conserved variant that differs in only a few amino acids, in this study semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of myosin heavy chain messenger RNAs (mRNAs) from the cross-striated tentacle and obliquely striated arm muscle fibres was conducted. This analysis showed that a previously reported alternatively spliced isoform of the squid myosin motor domain is present only in low abundance in both muscle types and therefore differential expression of the two myosins cannot explain the difference in contractile properties. It thus appears that modulation of the contractile properties of the musculature of squid and other cephalopods occurs primarily through variation in the arrangement and dimensions of the myofilaments.

Mutations within the human skeletal muscle alpha-actin gene cause three different skeletal muscle diseases. Functional studies of the mutant proteins are necessary to better understand the pathogenesis of these diseases, however, no satisfactory system for the expression of mutant muscle actin proteins has been available. We investigated the baculovirus expression vector system (BEVS) for the abundant production of both normal and mutant skeletal muscle alpha-actin. We show that non-mutated actin produced in the BEVS behaves similarly to native actin, as shown by DNase I affinity purification, Western blotting, and consecutive cycles of polymerisation and depolymerisation. Additionally, we demonstrate the production of mutant actin proteins in the BEVS, without detriment to the insect cells in which they are expressed. The BEVS therefore is the method of choice for studying mutant actin proteins causing human diseases.

Affinity purification of a phage-displayed library, expressing random peptide 12-mers at the N terminus of protein III, has identified 10 distinct novel sequences which bind troponin C specifically. The troponin C-selected peptides yield a consensus binding sequence of (V/L)(D/E)XLKXXLXXLA. Sequence comparison revealed as much as a 62.5% similarity between phiT5, the peptide sequence of the phage clone with the highest level of binding to troponin C, and the N-terminal region of troponin I isoforms. Biotinylated peptides corresponding to library-derived sequences and similar sequences from various isoforms of troponin I were synthesized shown to bind troponin C specifically. Alkaline phosphatase fusion proteins of two of the phage clone sequences bound troponin C specifically, and were specifically competed by both library-derived and native troponin I peptides. Measurement of equilibrium dissociation constants of the peptides by surface plasmon resonance yielded dissociation constants for troponin C as low as 0.43 microM for pT5; in contrast, dissociation constants for calmodulin were greater than 6 microM for all peptides studied. Nondenaturing polyacrylamide gel electrophoresis demonstrated that pT5 formed a stable complex with troponin C in the presence of calcium. We also found that the pT5 peptide inhibited the maximal calcium-activated tension of rabbit psoas muscle fibers.

New electron microscopic data provide direct evidence in support of the classical steric-blocking model for regulation of actin–myosin interactions by tropomyosin.

Sinusoidal analysis of the mechanochemical properties of skinned muscle fibers under conditions of maximal activation was applied to fibers from several rabbit skeletal muscles (psoas, tibialis anterior, extensor digitorum longus, diaphragm, soleus, semitendinosus). This investigation distinguished between two general classes of fibers, which on the basis of their myosin light chain complements could be classified as fast and slow. In fast fibers (e.g., psoas) we identified the presence of at least three exponential processes (A), (B), (C) of comparable magnitudes. In slow fibers (e.g., soleus) we identified the presence of at least four exponential processes (A)-(D) of very different magnitudes; magnitudes of processes (A) and (B) are very small compared with those of (C) and (D). The apparent rate constants are 8–29-fold slower in slow fibers. Because our sinusoidal characterization takes less than or equal to 22 s and does not involve chemical denaturation or other means of disruption of the myofilament lattice, it allows the different physiological classes of fibers to be characterized and then studied further by other techniques. The perfect correlation between physiological and molecular properties as assayed by gel electrophoresis after sinusoidal analysis demonstrates this and justifies its use in distinguishing between fiber types.

The early response to chronic low frequency stimulation is characterized by coordinate changes in fast thin filament and Z-line protein expression prior to the expression of slow contractile proteins. Within the first 3 weeks of intervention there is 1) a transition from expression of the fast troponin (Tn) Ts TnT1f and TnT2f to expression of TnT3f, 2) a parallel change in Z-line protein expression in which alpha-actinin1f/s becomes predominant, and 3) a small but significant increase in the levels of the beta-tropomyosin (Tm) subunit. The timing of these changes coincides with the conversion to thick Z-lines, and the kinetics of changes in fast TnT and alpha-actinin isoforms suggests that the expression of TnT3f with alpha-actinin1f/s and a combination of alpha beta and beta 2-Tm, which we have designated the TnT3f program, is coordinated. Because fast fibers expressing TnT3f with alpha beta and beta 2-Tm, like slow fibers, exhibit a more graded response to calcium (Schachat, F.H., Diamond, M.S., and Brandt, P.W. (1987) J. Mol. Biol., 198, 551-555), this change appears to be an adaptive response, illustrating the contribution of isoform diversity to the physiological plasticity of fast skeletal muscle and indicating that expression of the TnT3f program may be an intermediate phase in the conversion from a fast to a slow molecular phenotype.

Comparison of the myofibrillar proteins from several adult rabbit skeletal muscles has led to the identification of multiple forms of fast and slow troponin T. In Briggs et al. (Briggs, M. M., Klevit, R., and Schachat, F. H. (1984) J. Biol. Chem. 259, 10369-10375) two species of rabbit fast skeletal muscle troponin T (TnT), TnT1f and TnT2f, were characterized. Here, the distribution of these fast TnT species and the alpha- and beta- tropomyosin (Tm) subunits is characterized in fast muscles and in single muscle fibers. Evidence is also presented for two forms of slow skeletal muscle TnT. The presence of each fast TnT species is associated with the presence of a different Tm dimer: TnT1f with alpha beta-Tm and TnT2f with alpha 2-Tm. Histochemical analysis shows that expression of the fast TnT-Tm combinations is not due to differences in the distribution of fast-twitch glycolytic and fast-twitch oxidative-glycolytic fiber types. The absence of a correlation between histochemical typing and the composition of the thin filament Ca2+-regulatory complex is more apparent in individual fast muscle fibers where both fast TnT-Tm combinations appear to be expressed in a continuum. The implications of these observations for mammalian skeletal muscle fiber diversity are discussed.

The response of permeabilized rabbit fast skeletal muscle fibers to calcium is determined by the troponin T (TnT) and tropomyosin (Tm) isoforms they express. Fibers expressing primarily TnT 2f and α 2 Tm exhibit steeper pCa/tension relations than those in which either TnT 1f or TnT 3f and αβ Tm predominate. Troponin C extraction studies show that lower slopes do not result from a less concerted transition on the thin filament: the TnTm regulatory strand activates as a unit in all fast fibers. Because the TnT variants differ in their N-terminal segments, and this region overlaps adjacent Tms on the regulatory strand, we propose that both the end-to-end overlap of Tm and the effect of TnT on that interaction are the basis of the concerted transition of the regulatory strand to the active state that occurs in the presence of calcium. Moreover, the effect of different Tn-Tm combinations on the ratio of the affinities of TnC for calcium in the relaxed and active states appears to be a significant determinant of the contractile properties of fast fibers in vivo .

Extraction of as little as one troponin C per regulatory strand on a thin filament reduces the slope of the pCa/tension relation thus the regulatory units along a thin filament of rabbit psoas fibers are linked cooperatively so that a thin filament activates as a unit. This explains why the pCa/tension relation in skinned fibers has a slope much higher than can be expected by binding of Ca2+ to one regulatory unit (Brandt et al. 1980;Brandt et al. 1982). These results also show that troponin C is the only myfibrillar component responsible for calcium sensitivity in psoas muscle.

Calmodulin (CaM) and troponin C (TnC) are evolutionarily and structurally homologous, yet they are not functionally interchangeable. In particular, CaM cannot effectively substitute for TnC as an activator of skeletal muscle contraction. To determine if this is a consequence of CaM's weak association with troponin T and I or the result of a more fundamental mechanistic defect, we have used CaM and a CaM[TnC] chimera, CaM[3,4 TnC], that stably associates with the thin filament. Replacement of TnC with CaM or CaM[3,4 TnC] reveals that CaM-like molecules reduce the Ca2+-sensitivity and cooperativity of activation, as well as the maximal Ca2+-activated tension. These observations indicate that CaM-like molecules are unable to continuously maintain the activated state of the thin filament.

The relationship between histochemical fibre type and contractile protein expression was analysed in three rabbit skeletal muscles, the erector spinae, the plantaris and the diaphragm. A procedure for staining fibre bundles was developed using the same histochemical methods as those for typing fibres in cross-section. This allowed pretyped fibres to be selected and their molecular composition to be analysed by gel electrophoresis. The balance of expression of the two predominant fast troponin species, TnT1f and TnT2f, and alpha and beta tropomyosin subunits were studied in type IIA and IIB fast fibres. Type IIA fibres exhibited a restricted pattern of thin filament expression, exhibited TnT1f and both tropomyosin subunits in all three muscles. The expression in type IIB fibres, however, ranges from predominantly TnT2f and the alpha tropomyosin subunit in the erector spinae to TnT1f with both alpha and beta subunits in the diaphragm. These results indicate that there is not a simple one-to-one relationship between the fast muscle fibre subtypes and the expression of different thin filament protein isoforms.

In this paper we examine the role of two myosins in body-wall muscle cells of the nematode Caenorhabditis elegans. Large populations of nematodes are synchronized, and the synthesis and accumulation of myosin heavy chains and total protein are followed through postmitotic larval development. Growth is exponential with time for both the wild-type N2 and the body-wall muscledefective mutant E675, with a longer doubling time for the mutant. Utilizing the electrophoretic polymorphism of the E675 myosin heavy chains, we show that distinguishable classes of heavy chains accumulate differentially throughout development. Immunochemical measurements confirm a similar result in N2. Total myosin heavy chain accumulation is also quantitatively similar for the two strains. Myosin heavy chain relative synthetic rates as determined by pulse-labeling are constant throughout development and are equivalent for the two strains. The final fraction of accumulated unc -54 to total heavy chains of approximately 0.63 equals the constant synthetic fraction of approximately 0.62. Since myosin heavy chain accumulation and relative synthesis are equivalent, we conclude that the turnover of heavy chains is also similar in N2 and E675 despite the extensive structural and functional disruption within body-wall muscle cells of the latter strain. Since the accumulated fraction of unc -54 myosin heavy chains reaches a plateau at the constant synthetic fraction, myosin accumulation In the body-wall muscle cells may be attributed to a constant ratio of synthetic rates of the two body-wall myosin species. The coordinate synthesis of two myosins in the same body-wall muscle cells is discussed.

The properties of extraocular muscles (EOMs) are quite different from those of the trunk and limb. Here we show that there is a novel pattern of troponin T (TnT) expression in EOMs which most likely contributes to the fine control of ocular movement and may reflect their innervation by cranial motoneurons. Three regions of the muscle were analysed to distinguish the TnT isoforms present in the fast singly-innervated fibres from those in the multiply-innervated fibres. More than 95% of the TnT in the singly-innervated fibres is TnT3f, which exhibits the most graded response to changes in calcium concentration during activation (Schachat et al., J. molec. Biol. 198, 551-4). In multiply-innervated fibres, which exhibit tonic contractures, the slow troponin T TnT2s is expressed. While neither TnT3f nor TnT2s is unique to EOM, this pattern is unusual in two respects: first, both TnT3f and TnT2s are minor components of the trunk and limb musculature, and second, most muscles express several fast and both slow TnT species. Although EOM occupies a highly specialized physiological niche, its unusual physiology is not reflected in the presence of new TnT isoforms but in the expression of a different ratio of the known species of TnT.

The different isoforms of fast skeletal muscle troponin T (TnT) are generated by alternative splicing of several 5′ exons in the fast TnT gene. In rabbit skeletal muscle this process results in three major fast TnT species, TnT1f, TnT2f and TnT3f, that differ in a region of 30 to 40 amino acid residues near the N terminus. Differential expression of these three isoforms modulates the activation of the thin filament by calcium. To establish a basis for further structure-function studies, we have sequenced the N-terminal region of these proteins. TnT2f is the fast TnT sequenced by Pearlstone et al.. The larger species TnT1f contains six additional amino acid residues identical in sequence and position to those encoded by exon 4 in the rat fast skeletal muscle TnT gene. TnT3f also contains that sequence but lacks 17 amino acid residues spanning the region encoded by exons 6 and 7 of the rat gene. These three TnTs appear to be generated by discrete alternative splicing pathways, each differing by a single event. Comparison of these TnT sequences with those from chicken fast skeletal muscle and bovine heart shows that the splicing pattern resulting in the excision of exon 4 is evolutionarily conserved and leads to a more calcium-sensitive thin filament.

The extent and nature of fast troponin T (TnT) heterogeneity has been assessed in rabbit skeletal muscle. Previous studies identified two major fast TnT species (TnT1f and TnT2f), in the fast white muscle erector spinae, differing in their N-terminal cyanogen bromide (CNBr) fragments. Here a monoclonal antibody that recognizes a conserved region of TnT was used to characterize two additional TnT species (TnT3f and TnT4f) in the epaxial and limb musculature and a minor species (TnTcf) in craniofacial muscles. A combination of CNBr peptide mapping, immunoblotting and specific labelling of the N-terminus shows that these TnT species also differ in their N-terminal region. This observation is consistent with cDNA studies that predicted the N-terminal region is hypervariable. One additional species, a variant of TnT2f present in the tongue, was identified by two-dimensional gel electrophoresis. The limited number of TnT variants indicates that the full potential for heterogeneity inferred from the cDNA studies is not realized. This conclusion is supported by immunoblot analysis with a monoclonal antibody that recognizes an epitope in the hypervariable N-terminal region which is present in all variants of TnT1f and TnT2f but absent from the lower molecular weight species TnT3f and TnT4f.

The body-walls of Caenorhabditis elegans contain two different myosin heavy chains (Epstein, Waterston and Brenner, 1974) that associate to form at least two species of myosin (Schachat, Harris and Epstein, 1977a). To better define the distribution of these heavy chains in myosin molecules, we have characterized the myosin of C. elegans by immunochemical methods. Specific, precipitating anti-myosin antibody has been prepared in rabbits using highly purified nematode myosin as the immunogen. The difference in reactivity of the anti-myosin antibody with wild-type myosin containing both kinds of heavy chains (designated unc -54 and non- unc -54 heavy chains on the basis of genetic specification) and myosin from the mutant E190 that lacks unc -54 heavy chains Indicates that there are antigenic differences between myosin molecules containing unc -54 heavy chains and myosin molecules containing only non- unc -54 heavy chains. Antibody specific for the unc -54 myosin determinants has been prepared by the immunoadsorption of anti-myosin antibody with E190 myosin. This specific anti- unc -54 myosin antibody precipitates myosin that contains only unc -54 heavy chains. At the limits of resolution of our immunoprecipitation techniques, we could detect no heterodimeric myosin molecules containing both unc -54 and non- unc -54 heavy chains. The body-wall myosins of C. elegans therefore exist only as homodimers of either class of heavy chain. This specific anti- unc -54 myosin antibody promises to be a valuable tool in elucidating the role of two myosins in body-wall muscle and in molecular characterizations of mutant myosins in C. elegans. We report here the use of this antibody to detect antigenic differences between unc -54 myosin from the wild-type and the muscle mutant E675. In conjunction with the original anti-myosin antibody, other studies show that both unc -54 and non- unc -54 myosins exist within the same body-wall muscle cells (Mackenzie, Schachat and Epstein, 1978) and that both myosins are coordinately synthesized during muscle development in C. elegans (Garcea, Schachat and Epstein, 1978). We discuss the implications of the self-association of unc -54 and non- unc -54 myosin heavy chains into homodimeric myosins within the same body-wall muscles with respect to the assembly of thick filaments and their organization into a regular lattice.