Parallel inhibition of active force and relaxed fiber stiffness by caldesmon fragments at physiological ionic strength and temperature conditions: additional evidence that weak cross-bridge binding to actin is an essential intermediate for force generation.

Previously we showed that stiffness of relaxed fibers and active force generated in single skinned fibers of rabbit psoas muscle are inhibited in parallel by actin-binding fragments of caldesmon, an actin-associated protein of smooth muscle, under conditions in which a large fraction of cross-bridges is weakly attached to actin (ionic strength of 50 mM and temperature of 5 degrees C). These results suggested that weak cross-bridge attachment to actin is essential for force generation. The present study provides evidence that this is also true for physiological ionic strength (170 mM) at temperatures up to 30 degrees C, suggesting that weak cross-bridge binding to actin is generally required for force generation. In addition, we show that the inhibition of active force is not a result of changes in cross-bridge cycling kinetics but apparently results from selective inhibition of weak cross-bridge binding to actin. Together with our previous biochemical, mechanical, and structural studies, these findings support the proposal that weak cross-bridge attachment to actin is an essential intermediate on the path to force generation and are consistent with the concept that isometric force mainly results from an increase in strain of the attached cross-bridge as a result of a structural change associated with the transition from a weakly bound to a strongly bound actomyosin complex. This mechanism is different from the processes responsible for quick tension recovery that were proposed by Huxley and Simmons (Proposed mechanism of force generation in striated muscle. Nature. 233:533-538.) to represent the elementary mechanism of force generation.     

Domain mapping of chicken gizzard caldesmon

Limited proteolysis, affinity chromatography, and immunoblotting have been used to define the domains of chicken gizzard caldesmon, caldesmon120, that interact with calmodulin, F-actin, and a monoclonal antibody prepared using human platelet caldesmon. Treatment of caldesmon120 with chymotrypsin produces groups of fragments near 100, 80, 60, 38, and 20 kDa. Further digestion produces peptides between 40 and 50 kDa. The 100- and 80-kDa peptides cross-react with the monoclonal antibody; the smaller polypeptides do not. The kinetics of cleavage and the antibody studies indicate that the 38- and 80-kDa fragments are the two major pieces of the 120-kDa protein. The 38-kDa fragment, purified by high performance liquid chromatography, and several of its subfragments at 21 and 25 kDa sediment with F-actin, bind to calmodulin-Sepharose in the presence of Ca2+, and are displaced from F-actin by Ca2+-calmodulin. The 80-kDa fragments did not interact with F-actin or calmodulin. We have tentatively placed the 38-kDa fragment at the C-terminal using polyclonal antibodies selected against a beta-galactosidase-caldesmon120 fusion protein produced by a lambda gt11 lysogen. The 38-, 25-, and 21-kDa fragments cross-react with these antibodies; the 80- and 60-kDa fragments do not. Caldesmon77 from human platelets also cross-reacts with these selected antibodies. The results suggest that interacting calmodulin and F-actin binding sites are localized on a 38-kDa C-terminal fragment of caldesmon. The smallest subfragment of this peptide that binds to both F-actin and calmodulin-Sepharose is about 21 kDa. The monoclonal antibody epitope is tentatively localized near the N-terminal of caldesmon77 and must be within 50 kDa of the N-terminal on caldesmon120.     

Cross-bridge binding to actin and force generation in skinned fibers of the rabbit psoas muscle in the presence of antibody fragments against the N-terminus of actin.

To assess the significance of the NH2-terminus of actin for cross-bridge action in muscle, skinned fibers of rabbit psoas muscle were equilibrated with Fab fragments of antibodies directed against the first seven N-terminal residues of actin. With the antibody fragment, active force is more inhibited than relaxed fiber stiffness, or stiffness in rigor or in the presence of magnesium pyrophosphate. Inhibition of stiffness in rigor or with magnesium pyrophosphate does not necessarily indicate involvement of the NH2-terminus of actin in strong cross bridge binding to actin but may simply result from the large size of the Fab. At high Fab concentrations, active force is essentially abolished, whereas stiffness is still detectible under all conditions. Thus, complete inhibition of active force apparently is not due to interference with cross-bridge binding to actin but may result from the Fab-mimicking inhibition of the thin filament by Troponin-1 binding to the NH2-terminus of actin at low Ca2+. However, although Troponin-1 is released from the NH2-terminus at high Ca2+, the Fab is not, thus disallowing force generation upon increase in Ca2+. These data are consistent with involvement of the NH2-terminus of actin in both weak cross-bridge binding to actin and Ca2+ regulation of the thin filament.     

Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin

High-Mr caldesmon, which is involved in smooth muscle contraction, was phosphorylated by protein kinase C. By chymotryptic digestion, actin- and calmodulin-binding assays and immunoprecipitation with the antibody to the C-terminal 35-kDa fragment, we have identified that all phosphate groups are incorporated exclusively into this fragment, which is the functional domain for binding actin and calmodulin. Phosphorylation of high-Mr caldesmon and its C-terminal 35-kDa fragment reduced their binding abilities to both F-actin and calmodulin. Further, their inhibitory effects on the actin-activated ATPase activity of gizzard myosin were also reversed in proportion to the degree of phosphorylation. These results suggest that phosphorylation of high-Mr caldesmon by protein kinase C, which is restricted within the C-terminal 35-kDa domain, results in the modulation of its activity in the smooth muscle actin--myosin interaction.     

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.     

The smooth muscle cross-bridge cycle studied using sinusoidal length perturbations

The mechanical characteristics of smooth muscle can be broadly defined as either phasic, or fast contracting, and tonic, or slow contracting (, Pharmacol. Rev. 20:197-272). To determine if differences in the cross-bridge cycle and/or distribution of the cross-bridge states could contribute to differences in the mechanical properties of smooth muscle, we determined force and stiffness as a function of frequency in Triton-permeabilized strips of rabbit portal vein (phasic) and aorta (tonic). Permeabilized muscle strips were mounted between a piezoelectric length driver and a piezoresistive force transducer. Muscle length was oscillated from 1 to 100 Hz, and the stiffness was determined as a function of frequency from the resulting force response. During calcium activation (pCa 4, 5 mM MgATP), force and stiffness increased to steady-state levels consistent with the attachment of actively cycling cross-bridges. In smooth muscle, because the cross-bridge states involved in force production have yet to be elucidated, the effects of elevation of inorganic phosphate (P(i)) and MgADP on steady-state force and stiffness were examined. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 12 mM P(i), force and stiffness decreased proportionally, suggesting that cross-bridge attachment is associated with P(i) release. For the aorta, elevating P(i) decreased force more than stiffness, suggesting the existence of an attached, low-force actin-myosin-ADP- P(i) state. When portal vein strips were transferred from activating solution (pCa 4, 5 mM MgATP) to activating solution with 5 mM MgADP, force remained relatively constant, while stiffness decreased approximately 50%. For the aorta, elevating MgADP decreased force and stiffness proportionally, suggesting for tonic smooth muscle that a significant portion of force production is associated with ADP release. These data suggest that in the portal vein, force is produced either concurrently with or after P(i) release but before MgADP release, whereas in aorta, MgADP release is associated with a portion of the cross-bridge powerstroke. These differences in cross-bridge properties could contribute to the mechanical differences in properties of phasic and tonic smooth muscle.     

Critical dependence of calcium-activated force on width in highly compressed skinned fibers of the frog.

Force development by skinned frog semitendinosus fibers was studied at various levels of lateral compression to compare the results with intact fibers and to evaluate the limits on cross-bridge movements during isometric contraction. The skinned fibers were compressed osmotically using a high molecular weight polymer, dextran T500. Ca-activated force remained constant down to 58% of the fiber width (w0) after skinning, corresponding to a nearly twofold change in separation between the thin and thick filaments in the myofilament lattice. This agrees with the earlier result on intact fibers, and gives additional evidence that the cross-bridge mechanism for force generation is relatively insensitive to large changes in interfilament separation. Further compression, below 0.58 w0, produced a sharp drop in force, and the force was practically zero at a fiber width of 50%. The effect at high compression was the same at all pCa's, which indicates that the Ca sensitivity of the myofilaments is unaffected by radial compression. The stiffness of the fiber remained high in rigor in the presence of dextran, which indicates that the rigor cross-bridge attachment is not inhibited, and actually may be improved, with decreases in the interfilament space. Also, the drop in active force with the highest compression was similar when the compressed fibers were put in rigor before contraction, which suggests that the force drop also was not due to a hindrance to cross-bridge attachment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Force enhancement without changes in cross-bridge turnover kinetics: the effect of EMD 57033.

The thiadiazinon derivative EMD 57033 has been found previously in cardiac muscle to increase isometric force generation without a proportional increase in fiber ATPase, thus causing a reduction in tension cost. To analyze the mechanism by which EMD 57033 affects the contractile system, we studied its effects on isometric force, isometric fiber ATPase, the rate constant of force redevelopment (k(redev)), active fiber stiffness, and its effect on Fo, which is the force contribution of a cross-bridge in the force-generating states. We used chemically skinned fibers of the rabbit psoas muscle. It was found that with 50 microM EMD 57033, isometric force increases by more than 50%, whereas Kredev, active stiffness, and isometric fiber ATPase increase by at most 10%. The results show that EMD 57033 causes no changes in cross-bridge turnover kinetics and no changes in active fiber stiffness that would result in a large enough increase in occupancy of the force-generating states to account for the increase in active force. However, plots of force versus length change recorded during stretches and releases (T plots) indicate that in the presence of EMD 57033 the y(o) value (x axis intercept) for the cross-bridges becomes more negative while its absolute value increases. This might suggest a larger cross-bridge strain as the basis for increased active force. Analysis of T plots with and without EMD 57033 shows that the increase in cross-bridge strain is not due to a redistribution of cross-bridges among different force-generating states favoring states of larger strain. Instead, it reflects an increased cross-bridge strain in the main force-generating state. The direct effect of EMD 57033 on the force contribution of cross-bridges in the force-generating states represents an alternative mechanism for a positive inotropic intervention.     

Production of chymotrypsin-resistant Bacillus thuringiensis Cry2Aa1 delta-endotoxin by protein engineering.

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when (125)I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Conformational transitions in the myosin head induced by temperature, nucleotide and actin. Studies on subfragment-1 of myosins from rabbit and frog fast skeletal muscle with a limited proteolysis method

Tryptic digestion patterns reveal a close similarity of the substructure of frog subfragment-1 (S1) to that established for rabbit S1. The 97-kDa heavy chain of chymotryptic S1 of frog myosin is preferentially cleaved into three fragments with apparent molecular masses of 29 kDa, 49 kDa and 20 kDa. These fragments correspond to the 27-kDa, 50-kDa and 20-kDa fragments of rabbit S1, respectively; this is indicated by the sequence of their appearance during digestion, by the suppression by actin of the generation of the 49-kDa and 20-kDa peptides, and by a nucleotide-promoted cleavage of the 29-kDa peptide to a 24-kDa fragment and the 49-kDa peptide to a 44-kDa fragment, analogous to the nucleotide-promoted cleavage of the 27-kDa and 50-kDa fragments of rabbit S1 to the 22-kDa and 45-kDa peptides. The same changes in the digestion patterns as those produced by the presence of nucleotide (ATP or its beta,gamma-imido analog AdoP P[NH]P) at 25 degrees C were observed when the digestion was carried out at 0 degrees C in the absence of nucleotide. The low-temperature-induced changes were particularly well seen in the preparations from frog myosin. The presence of ATP or AdoP P[NH]P at 0 degrees C enhanced, whereas the complex formation with actin prevented, the low-temperature-induced changes. The results are consistent with there being two fundamental conformational states of the myosin head in an equilibrium that is dependent on the temperature, the nucleotide bound at the active site, and the presence or absence of actin.     

Tryptic fragments of the Escherichia coli DNA gyrase A protein

Treatment of the Escherichia coli DNA gyrase A protein with trypsin generates two large fragments which are stable to further digestion. The molecular masses of these fragments are 64 and 33 kDa, and they are shown to be derived from the N terminus and the C terminus of the A protein, respectively. These fragments could represent structural and/or functional domains within the A subunit of DNA gyrase. The trypsin-cleaved A protein (A'), in combination with the B subunit of gyrase, can support ATP-dependent supercoiling of relaxed DNA and other reactions of DNA gyrase. The isolated 64-kDa fragment will also catalyse DNA supercoiling in the presence of the B protein, but the 33-kDa fragment shows no enzymic activities. We conclude that the N-terminal 64-kDa fragment represents the DNA breakage/reunion domain of the A protein, while the 33-kDa fragment may contribute to the stability of the gyrase-DNA complex.     

Production of Chymotrypsin-Resistant Bacillus thuringiensis Cry2Aa1 δ-Endotoxin by Protein Engineering

Cleavage of the Cry2Aa1 protoxin (molecular mass, 63 kDa) from Bacillus thuringiensis by midgut juice of gypsy moth (Lymantria dispar) larvae resulted in two major protein fragments: a 58-kDa fragment which was highly toxic to the insect and a 49-kDa fragment which was not toxic. In the midgut juice, the protoxin was processed into a 58-kDa toxin within 1 min, but after digestion for 1 h, the 58-kDa fragment was further cleaved within domain I, resulting in the protease-resistant 49-kDa fragment. Both the 58-kDa and nontoxic 49-kDa fragments were also found in vivo when ¹²⁵I-labeled toxin was fed to the insects. N-terminal sequencing revealed that the protease cleavage sites are at the C termini of Tyr49 and Leu144 for the active fragment and the smaller fragment, respectively. To prevent the production of the nontoxic fragment during midgut processing, five mutant proteins were constructed by replacing Leu144 of the toxin with Asp (L144D), Ala (L144A), Gly (L144G), His (L144H), or Val (L144V) by using a pair of complementary mutagenic oligonucleotides in PCR. All of the mutant proteins were highly resistant to the midgut proteases and chymotrypsin. Digestion of the mutant proteins by insect midgut extract and chymotrypsin produced only the active 58-kDa fragment, except that L144H was partially cleaved at residue 144.     

Phosphorylation of caldesmon prevents its interaction with smooth muscle myosin

Caldesmon is known to bind to smooth muscle myosin. Ca2+/calmodulin-dependent phosphorylation of caldesmon completely blocks its interaction with myosin. Cleavage of caldesmon at its 2 cysteine residues by 2-nitro-5-thiocyanobenzoic acid (NTCB) occurs initially at one site to yield 108-kDa and 21.2-kDa peptides and subsequently at the second site within the 108-kDa peptide to yield 85-kDa and 23.5-kDa fragments. The 23.5-kDa peptide retains the ability to bind to myosin. The N-terminal (95 kDa) and C-terminal (42 kDa) chymotryptic peptides of caldesmon were isolated and digested with NTCB: the C-terminal actin- and calmodulin-binding peptide was not cleaved, indicating that it does not contain either of the cysteine residues, whereas the 95-kDa N-terminal peptide was cleaved at two sites to yield 56-kDa, 23.5-kDa, and 21.2-kDa fragments. The arrangement of NTCB fragments in caldesmon is, therefore: 21.2 kDa/23.5 kDa/85 kDa from N to C terminus. Digestion of phosphorylated caldesmon with NTCB suggested a single phosphorylation site in the 21.2-kDa peptide and three sites in the 23.5-kDa peptide. These results lead to the development of a model whereby caldesmon may cross-link actin to myosin and such cross-linking is blocked by phosphorylation of caldesmon. This mechanism may explain the formation of reversible "latch bridges" which permit force maintenance at low levels of myosin phosphorylation in intact smooth muscles.     

Thixotropy and Rheopexy of Muscle Fibers Probed Using Sinusoidal Oscillations

Length changes of muscle fibers have previously been shown to result in a temporary reduction in fiber stiffness that is referred to as thixotropy. Understanding the mechanism of this thixotropy is important to our understanding of muscle function since there are many instances in which muscle is subjected to repeated patterns of lengthening and shortening. By applying sinusoidal length changes to one end of single permeabilized muscle fibers and measuring the force response at the opposite end, we studied the history-dependent stiffness of both relaxed and activated muscle fibers. For length change oscillations greater than 1 Hz, we observed thixotropic behavior of activated fibers. Treatment of these fibers with EDTA and blebbistatin, which inhibits myosin-actin interactions, quashed this effect, suggesting that the mechanism of muscle fiber thixotropy is cross-bridge dependent. We modeled a half-sarcomere experiencing sinusoidal length changes, and our simulations suggest that thixotropy could arise from force-dependent cross-bridge kinetics. Surprisingly, we also observed that, for length change oscillations less than 1 Hz, the muscle fiber exhibited rheopexy. In other words, the stiffness of the fiber increased in response to the length changes. Blebbistatin and EDTA did not disrupt the rheopectic behavior, suggesting that a non-cross-bridge mechanism contributes to this phenomenon.     

Suppression of muscle contraction by vanadate. Mechanical and ligand binding studies on glycerol-extracted rabbit fibers

The suppression of tension development by orthovanadate (Vi) was studied in mechanical experiments and by measuring the binding of radioactive Vi and nucleotides to glycerol-extracted rabbit muscle fibers. During active contractions, Vi bound to the cross-bridges and suppressed tension with an apparent second-order rate constant of 1.34 X 10(3) M-1s-1. The half-saturation concentration for tension suppression was 94 microM Vi. The incubation of fibers in Vi relaxing or rigor solutions prior to initiation of active contractions had little effect on the initial rise of active tension. The addition of adenosine diphosphate (ADP) and Vi to fibers in rigor did not cause relaxation. Suppression of tension only developed during cross-bridge cycling. After slow relaxation from rigor in 1 mM Vi and low (50 microM) MgATP concentration (0 Ca2+), radioactive Vi and ADP were trapped within the fiber. This finding indicated the formation of a stable myosin X ADP X Vi complex, as has been reported in biochemical experiments with isolated myosin. Vi and ADP trapped within the fibers were released only by subsequent cross-bridge attachment. Vi and ADP were preferentially trapped under conditions of cross-bridge cycling in the presence of ATP rather than in relaxed fibers or in rigor with ADP. These results indicate that in the normal cross-bridge cycle, inorganic phosphate (Pi) is released from actomyosin before ADP. The resulting actomyosin X ADP intermediate can bind Vi and Pi. This intermediate probably supports force. Vi behaves as a close analogue of Pi in muscle fibers, as it does with isolated actomyosin.     

Proteolytic fragmentation of brain myosin and localisation of the heavy-chain phosphorylation site

The heavy chains and the 19-kDa and 20-kDa light chains of bovine brain myosin can by phosphorylated. To localise the site of heavy-chain phosphorylation, the myosin was initially subjected to digestion with chymotrypsin and papain under a variety of conditions and the fragments thus produced were identified. Irrespective of the ionic strength, i.e. whether the myosin was monomeric or filamentous, chymotryptic digestion produced two major fragments of 68 kDa and 140 kDa; the 140-kDa fragment was further digested by papain to yield a 120-kDa and a 23-kDa fragment. These fragments were characterised by (a) a gel overlay technique using 125I-labelled light chains, which showed that the 140-kDa and 23-kDa polypeptides contain the light-chain-binding sites; (b) using myosin photoaffinity labelled at the active site with [3H]UTP, which showed that the 68-kDa fragment contained the catalytic site, and (c) electron microscopy, using rotary shadowing and negative-staining techniques, which demonstrated that after chymotryptic digestion the myosin head remains attached to the tail whereas on papain digestion isolated heads and tails were observed. Thus the 120-kDa polypeptide derived from the 140-kDa fragment is the tail of the myosin, and the 68-kDa fragment containing the catalytic site and the 23-kDa fragment, with the light-chain-binding sites, form the head (S1) portion of the myosin. When [32P]-phosphorylated brain myosin was digested with chymotrypsin and papain it was shown that the heavy-chain phosphorylation site is located in a 5-kDa peptide at the C-terminal end of the heavy chain, i.e. the end of the myosin tail. Using hydrodynamic and electron microscopic techniques, no significant effect of either light-chain or heavy-chain phosphorylation on the stability of brain myosin filaments was observed, even in the presence of MgATP. Brain myosin filaments appear to be more stable than those of other non-muscle myosins. Light-chain phosphorylation did, however, have an effect on the conformation of brain myosin, for example in the presence of MgATP non-phosphorylated myosin molecules were induced to fold into a very compact folded state.     

Muscle force and stiffness during activation and relaxation. Implications for the actomyosin ATPase

Isolated skinned frog skeletal muscle fibers were activated (increasing [Ca2+]) and then relaxed (decreasing [Ca2+]) with solution changes, and muscle force and stiffness were recorded during the steady state. To investigate the actomyosin cycle, the biochemical species were changed (lowering [MgATP] and elevating [H2PO4-]) to populate different states in the actomyosin ATPase cycle. In solutions with 200 microM [MgATP], compared with physiological [MgATP], the slope of the plot of relative steady state muscle force vs. stiffness was decreased. At low [MgATP], cross-bridge dissociation from actin should be reduced, increasing the population of the last cross-bridge state before dissociation. These data imply that the last cross-bridge state before dissociation could be an attached low-force-producing or non-force-producing state. In solutions with 10 mM total Pi, compared to normal levels of MgATP, the maximally activated muscle force was reduced more than muscle stiffness, and the slope of the plot of relative steady state muscle force vs. stiffness was reduced. Assuming that in elevated Pi, Pi release from the cross-bridge is reversed, the state(s) before Pi release would be populated. These data are consistent with the conclusion that the cross-bridges are strongly bound to actin before Pi release. In addition, if Ca2+ activates the ATPase by allowing for the strong attachment of the myosin to actin in an A.M.ADP.Pi state, it could do so before Pi release. The calcium sensitivity of muscle force and stiffness in solutions with 4 mM [MgATP] was bracketed by that measured in solutions with 200 microM [MgATP], where muscle force and stiffness were more sensitive to calcium, and 10 mM total Pi, where muscle force and stiffness were less sensitive to calcium. The changes in calcium sensitivity were explained using a model in which force-producing and rigor cross-bridges can affect Ca2+ binding or promote the attachment of other cross-bridges to alter calcium sensitivity.     

Caldesmon inhibits formation of strongly bound myosin cross-bridges and activates an ability of weakly bound cross-bridges to transform actin monomers to the off-conformation

The effect of caldesmon and its actin-binding C-terminal 35 kDa fragment on conformational alterations of actin in a muscle fiber at relaxation, rigor and at simulation of strong and weak binding of myosin heads to actin was studied by polarizational fluorimetry technique. The strong and weak binding forms were mimicked during binding of F-actin of ghost muscle fibers to myosin subfragment-1 modified with NEM (NEM-S1) or pPDM (pPDM-S1), respectively. As a test for alterations in actin conformation, changes in orientation and mobility of a fluorescent probe, TRITC-phalloidin, bound specifically to F-actin were used. The results obtained have shown that during transition of the muscle fiber from the relaxed state into the rigor and during binding of actin filaments to NEM-S1, changes of polarization parameters take place, which are characteristic of formation between actin and myosin of the strong binding and of transformation of actin subunits from the "turned-off" (inactive) to the "turned-on" (active) conformation. Binding of pPDM-S1 to actin and relaxation of the muscle fiber are accompanied, on the contrary, by the changes of orientation and of the fluorescent probe mobility, which are typical of formation of the weak ("non-force-producing") form of actin-myosin binding and of transformation of actin subunits from the active conformation into the inactive one. Caldesmon and its C-terminal fragment markedly inhibit formation of the strong binding at rigor and activate transition of actin monomers to the switched off conformation at relaxation of muscle fiber. In parallel experiments, these regulatory proteins have been shown to inhibit an active force developed at the transition of a muscle fiber from relaxation to rigor. Besides, caldesmon and its fragment decrease the rate of actin filament sliding over myosin in an in vitro motility assay. Caldesmon is suggested to regulate the smooth muscle contraction in an allosterical manner. The alterations in actin conformation inhibit formation of strong binding of myosin cross bridges to actin and activate the ability of weakly bound cross bridges to switch actin monomers from the "on" to the "off" conformation.     

Distribution of secondary structure along the fibronectin molecule

30-kDa, 50-kDa and 70-kDa gelatin-binding, 60-kDa central and 60-65-kDa heparin-binding fragments were produced by trypsin digestion of fibronectin. The secondary structure of the fragments was studied by circular dichroism and quantitative infrared spectroscopy. The structure of the 70-kDa gelatin-binding, 60-kDa central and 60-65-kDa heparin-binding fragments in solution appeared to be very close to that in the intact fibronectin. The content of the antiparallel beta-form, the only element of the secondary structure in all the fragments studied, was shown to be 30-35%.     

The frequency response of smooth muscle stiffness during Ca2+-activated contraction

To investigate the mechanism of smooth muscle contraction, the frequency response of the muscle stiffness of single beta-escin permeabilized smooth muscle cells in the relaxed state was studied. Also, the response was continuously monitored for 3 min from the beginning of the exchange of relaxing solution to activating solution, and then at 5-min intervals for up to 20 min. The frequency response (30 Hz bandwidth, 0.33 Hz (or 0.2 Hz) resolution) was calculated from the Fourier-transformed force and length sampled during a 3-s (or 5-s) constant-amplitude length perturbation of increasing-frequency (1-32 Hz) sine waves. In the relaxed state, a large negative phase angle was observed, which suggests the existence of attached energy generating cross-bridges. As the activation progressed, the muscle stiffness and phase angle steadily increased; these increases gradually extended to higher frequencies, and reached a steady state by 100 s after activation or approximately 40 s after stiffness began to increase. The results suggest that a fixed distribution of cross-bridge states was reached after 40 s of Ca2+ activation and the cross-bridge cycling rate did not change during the period of force maintenance.