Rabbit Psoas Muscle Research Articles

Cross-Bridge Attachment during High-Speed Active Shortening of Skinned Fibers of the Rabbit Psoas Muscle: Implications for Cross-Bridge Action during Maximum Velocity of Filament Sliding

To characterize the kinetics of cross-bridge attachment to actin during unloaded contraction (maximum velocity of filament sliding), ramp-shaped stretches with different stretch-velocities (2–40,000 nm per half-sarcomere per s) were applied to actively contracting skinned fibers of the rabbit psoas muscle. Apparent fiber stiffness observed during such stretches was plotted versus the speed of the imposed stretch (stiffness-speed relation) to derive the rate constants for cross-bridge dissociation from actin. The stiffness-speed relation obtained for unloaded shortening conditions was shifted by about two orders of magnitude to faster stretch velocities compared to isometric conditions and was almost identical to the stiffness-speed relation observed in the presence of MgATP γS at high Ca 2+ concentrations, i.e., under conditions where cross-bridges are weakly attached to the fully Ca 2+ activated thin filaments. These data together with several control experiments suggest that, in contrast to previous assumptions, most of the fiber stiffness observed during high-speed shortening results from weak cross-bridge attachment to actin. The fraction of strongly attached cross-bridges during unloaded shortening appears to be as low as some 1–5% of the fraction present during isometric contraction. This is about an order of magnitude less than previous estimates in which contribution of weak cross-bridge attachment to observed fiber stiffness was not considered. Our findings imply that 1) the interaction distance of strongly attached cross-bridges during high-speed shortening is well within the range consistent with conventional cross-bridge models, i.e., that no repetitive power strokes need to be assumed, and 2) that a significant part of the negative forces that limit the maximum speed of filament sliding might originate from weak cross-bridge interactions with actin.

Effect of Oxygen Free Radicals on Myosin in Muscle Fibres. A DSC and EPR study

Differential scanning calorimetry (DSC) and electron paramagnetic resonance spectroscopy (EPR, both conventional and saturation transfer EPR) were used to study the motional dynamics and segmental flexibility of myosin in muscle fibres in the presence of free radical generating system. Muscle fibre bundles isolated from psoas muscle of rabbit were spin-labelled with maleimide-and isothiocyanate-based probe molecules at the reactive sulfhydryl sites (Cys-707) of the motor domain. In the presence of hydroxyl free radicals the spectral intensity of the maleimide probe molecules decreased with time following a single exponential curve. MgADP and MgATP plus orthovanadate that produce flexibility changes in the multisubunit structure of myosin enhanced the reduction of the attached nitroxide molecules in free radical generating system. The analysis of the EPR spectra of spin-labelled and oriented fibres showed that the narrow distribution of spin labels changed in the presence of hydroxyl free radicals. Spectrum analysis by computer subtraction showed that short irradiation by UV light resulted in the enhancement of the ordered population at the expense of the disordered population. This suggests a transition of myosin heads from weak-binding state into strong-binding state. DSC measurements performed on calf cardiac myosin resulted in two main transitions at 49.4 and 54.1°C, respectively. Addition of MgADP produced a decrease of the 49.4°C transition, whereas a shift towards higher temperature was detected at the 54.1°C transition. It shows that there is an inter-site communication between the domains of the myosin. Hydroxyl free radicals induced further shifts of the transition temperatures and affected the width of the heat absorption curves.

A flash photolysis fluorescence/light scattering apparatus for use with sub microgram quantities of muscle proteins.

Transient kinetic methods such as stopped flow and quenched flow have been used to elucidate many of the fundamental features of the molecular interactions which underlie muscle contraction. However, these methods traditionally require relatively large amounts of protein (10(-3) g) and so have been used most effectively for the proteins purified from bulk muscle tissue of large animals or where the proteins can be expressed in large amounts (e.g.. Dictyostelium). We have investigated the use of flash photolysis of an inert precursor of ATP (cATP) to initiate the dissociation of acto.S1 and acto.myosin and the subsequent ATP turnover reaction. Using a sample volume of 10 microl we show that a significant amount of information on the transient and steady-state kinetics of the system can be obtained from a sample containing just 50 nM of acto.myosin or acto.S1 complex in solution. Therefore in presence of excess of one protein component the measurements require only 250 ng myosin, 62 ng S1 or 25 ng actin. This is therefore the method of choice for kinetic analysis of acto.myosins which are only available in microgram quantities. We report for the first time the determination of the second order rate constant of ATP-induced dissociation of actin from the myosin extracted from a single fibre from a rabbit psoas muscle.

Contractility of Single Myofibrils of Rabbit Skeletal Muscle Studied at Various MgATP Concentrations.

A novel experimental method was developed to study the contractility of single myofibrils of skeletal muscle. Single myofibrils (ca. 1 microm in diameter) prepared from glycerinated rabbit psoas muscle were suspended between rigid and flexible microneedles by the entwining method. The length changes of the preparations applied via the rigid microneedle by an actuator and the force produced were measured by photo-electrically detecting the nanometer deflections of the flexible microneedle. Single myofibril preparations maintained uniform sarcomere striations during contraction-relaxation cycles. The isometric force produced, the velocity of unloaded shortening, and the force-velocity relationship of single myofibrils were investigated at various MgATP concentrations. The contractility of single myofibrils thus obtained in the absence of ATP regenerative systems was essentially the same as that of skinned muscle fibers under comparable conditions in the presence of ATP regenerative systems. Thus, it was found that (1) the present experimental method is useful for studying the contractility of single myofibrils, and (2) in single myofibril preparations, the MgATP concentration at actomyosin sites is well equilibrated with that in bathing solutions.

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg 2+ or MgADP or MgATP. The entire myosin molecule forms head–head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95°. Assuming that the length of the globular part of S1 is ≈12 nm and that the S1/S2 joint (lever arm ≈7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of ≈18 nm, close to the ≈16–20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament–filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature (≈22°C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg 2+ or 0.5 mM MgATP and at low temperature (≈0°C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature (≈0°C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament–filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).

Thin Filament Activation Probed by Fluorescence of N-((2-(Iodoacetoxy)ethyl)- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-Labeled Troponin I Incorporated into Skinned Fibers of Rabbit Psoas Muscle

A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label ( N-((2-(iodoacetoxy)ethyl)- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole, IANBD) on Cys 133 of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (Trybus and Taylor, 1980. Proc. Natl. Acad. Sci. USA. 77:7209–7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca 2+ or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca 2+ is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca 2+ concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca 2+ concentrations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.

The M·ADP·P i State Is Required for Helical Order in the Thick Filaments of Skeletal Muscle

The thick filaments of mammalian and avian skeletal muscle fibers are disordered at low temperature, but become increasingly ordered into an helical structure as the temperature is raised. Wray and colleagues (Schlichting, I., and J. Wray. 1986. J. Muscle Res. Cell Motil. 7:79; Wray, J., R. S. Goody, and K. Holmes. 1986. Adv. Exp. Med. Biol. 226:49–59) interpreted the transition as reflecting a coupling between nucleotide state and global conformation with M·ATP (disordered) being favored at 0°C and M·ADP·P i (ordered) at 20°C. However, hitherto this has been limited to a qualitative correlation and the biochemical state of the myosin heads required to obtain the helical array has not been unequivocally identified. In the present study we have critically tested whether the helical arrangement of the myosin heads requires the M·ADP·P i state. X-ray diffraction patterns were recorded from skinned rabbit psoas muscle fiber bundles stretched to non-overlap to avoid complications due to interaction with actin. The effect of temperature on the intensities of the myosin-based layer lines and on the phosphate burst of myosin hydrolyzing ATP in solution were examined under closely matched conditions. The results showed that the fraction of myosin mass in the helix closely followed that of the fraction of myosin in the M·ADP·P i state. Similar results were found by using a series of nucleoside triphosphates, including CTP and GTP. In addition, fibers treated by N-phenylmaleimide (Barnett, V. A., A. Ehrlich, and M. Schoenberg. 1992. Biophys. J. 61:358–367) so that the myosin was exclusively in the M·ATP state revealed no helical order. Diffraction patterns from muscle fibers in nucleotide-free and in ADP-containing solutions did not show helical structure. All these confirmed that in the presence of nucleotides, the M·NDP·P i state is required for helical order. We also found that the spacing of the third meridional reflection of the thick filament is linked to the helical order. The spacing in the ordered M·NDP·P i state is 143.4 Å, but in the disordered state, it is 144.2 Å. This may be explained by the different interference functions for the myosin heads and the thick filament backbone.

Kinetics of Thin Filament Activation Probed by Fluorescence of N-((2-(Iodoacetoxy)ethyl)- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-Labeled Troponin I Incorporated into Skinned Fibers of Rabbit Psoas Muscle: Implications for Regulation of Muscle Contraction

Making use of troponin with fluorescently labeled troponin I subunit ( N-((2-(iodoacetoxy)ethyl)- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (Trybus and Taylor, 1980. Proc. Natl. Acad. Sci. 77:7209–7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (Brenner et al., 1999. Biophys. J. 77:000–000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca 2+ concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca 2+ concentrations. At all Ca 2+ concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca 2+ is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca 2+-dependent equilibration among different states of the thin filament can fully account for the Ca 2+ dependence of force redevelopment and the fluorescence changes described in this study.

Effects of inorganic phosphate on endothermic force generation in muscle.

Using a rapid (ca. 0.2 ms) laser temperature jump technique, the rate of endothermic force generation was examined in single-skinned (rabbit psoas) muscle fibres when they were exposed to different levels of inorganic phosphate (a product released during ATP hydrolysis in active muscle). The steady force is reduced by increased phosphate but the apparent rate constant of force generation induced by a standard temperature jump (from ca. 9 degrees C to ca. 12 degrees C) increases two- to threefold when the phosphate added is increased from zero to ca. 25 mM. The increase in the apparent rate constant also exhibits saturation at higher phosphate levels and the relation is hyperbolic. Detailed examination of the data, particularly in relation to our pressure release experiments, leads to a scheme for the molecular steps involved in phosphate release and force generation in active muscle fibres, where phosphate release from attached cross-bridges involves three reversible and sequentially faster molecular steps. Step one is a moderately slow, pre-force generation step that probably represents a transition of cross-bridges from non-specific to stereospecific attached states. Step two is moderately fast and represents endothermic cross-bridge force generation (temperature sensitive) and step three is a very rapid phosphate release. Such a scheme accommodates findings from a variety of different studies, including pressure perturbation experiments and other studies where the effect of phosphate on muscle force was studied.

The efficiency of contraction in rabbit skeletal muscle fibres, determined from the rate of release of inorganic phosphate.

1. The relationship between mechanical power output and the rate of ATP hydrolysis was investigated in segments of permeabilized fibres isolated from rabbit psoas muscle. 2. Contractions were elicited at 12 degrees C by photolytic release of ATP from the P3 -1-(2-nitrophenyl) ester of ATP (NPE-caged ATP). Inorganic phosphate (Pi) release was measured by a fluorescence method using a coumarin-labelled phosphate binding protein. Force and sarcomere length were also monitored. 3. ATPase activity was determined from the rate of appearance of Pi during each phase of contraction. The ATPase rate was 10.3 s-1 immediately following release of ATP and 5. 1 s-1 during the isometric phase prior to the applied shortening. It rose hyperbolically with shortening velocity, reaching 18.5 s-1 at a maximal shortening velocity > 1 ML s-1 (muscle lengths s-1). 4. Sarcomeres shortened at 0.09 ML s-1 immediately following the photolytic release of ATP and at 0.04 ML s-1 prior to the period of applied shortening. The high initial ATPase rate may be largely attributed to initial sarcomere shortening. 5. During shortening, maximal power output was 28 W l-1. Assuming the free energy of hydrolysis is 50 kJ mol-1, the efficiency of contraction was calculated from the power output at each shortening velocity. The maximum efficiency was 0.36 at a shortening velocity of 0.27 ML s-1, corresponding to a force level 51 % of that in the isometric state. 6. At the maximal shortening velocity, only 10 % of the myosin heads are attached to the thin filaments at any one time.

The Effect of Thin Filament Activation on the Attachment of Weak Binding Cross-Bridges: A Two-Dimensional X-Ray Diffraction Study on Single Muscle Fibers

To study possible structural changes in weak cross-bridge attachment to actin upon activation of the thin filament, two-dimensional (2D) x-ray diffraction patterns of skinned fibers from rabbit psoas muscle were recorded at low and high calcium concentration in the presence of saturating concentrations of MgATP γS, a nucleotide analog for weak binding states. We also studied 2D x-ray diffraction patterns recorded under relaxing conditions at an ionic strength above and below 50 mM, because it had been proposed from solution studies that reducing ionic strength below 50 mM also induces activation of the thin filament. For this project a novel preparation had to be established that allows recording of 2D x-ray diffraction patterns from single muscle fibers instead of natural fiber bundles. This was required to minimize substrate depletion or product accumulation within the fibers. When the calcium concentration was raised, the diffraction patterns recorded with MgATP γS revealed small changes in meridional reflections and layer line intensities that could be attributed in part to the effects of calcium binding to the thin filament (increase in I 380, decrease in first actin layer line intensity, increase in I 59) and in part to small structural changes of weakly attached cross-bridges (e.g., increase in I 143 and I 72). Calcium-induced small-scale structural rearrangements of cross-bridges weakly attached to actin in the presence of MgATP γS are consistent with our previous observation of reduced rate constants for attachment and detachment of cross-bridges with MgATP γS at high calcium. Yet, no evidence was found that weakly attached cross-bridges change their mode of attachment toward a stereospecific conformation when the actin filament is activated by adding calcium. Similarly, reducing ionic strength to less than 50 mM does not induce a transition from nonstereospecific to stereospecific attachment.

Ca 2+ and Cross-Bridge-Induced Changes in Troponin C in Skinned Skeletal Muscle Fibers: Effects of Force Inhibition

Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca 2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5′ isomer of iodoacetamidotetramethylrhodamine (5′IATR), attached to Cys 98 (sTnC-5′ATR), in sTnC-5′ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca 2+ and cross-bridge binding on sTnC structure, maximum Ca 2+-activated force was inhibited with 0.5 mM AlF 4 − or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca 2+ dependence of force and dichroism. Dichroism was 0.08 ± 0.01 (± SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 ± 0.002 (± SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca 2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca 2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF 4 − decreased force to 0.04 ± 0.01 of maximum (± SEM, n = 3), and dichroism was 0.04 ± 0.03 (± SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 ± 0.05, ± SEM; n = 3). Inhibition of force with AlF 4 − also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-( N-(iodoacetoxy)ethyl- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 μm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 ± 0.03 (± SEM, n = 5) of the value at pCa 9.2, and force was 0.51 ± 0.04 (± SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 ± 0.03 (± SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 ± 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca 2+].

Contractile properties of rabbit psoas muscle fibres inhibited by beryllium fluoride.

The structure of truncated, recombinant Dictyostelium myosin motor domain complexed with Mg.ADP and slowly dissociating analogues of Pi has previously been characterized as two main states (S1-MgADP plus BeFx vs. A1F4- or Vi). The BeFx bound state is thought to mimic the weak actin-binding M.ATP complex, while the states with A1F4- or Vi bound mimic the M.ADP.Pi state. While the effects of A1F4- and Vi on fibre mechanics have been previously described (Chase et al., 1994, 1993), the effects of BeFx have not been characterized in detail. At pCa 4.5 (12 degrees C), we measured (i) steady-state isometric tension, (ii) stiffness (KS; 1 kHz sinusoids), and (iii) unloaded shortening velocity (Vu; slack test) in single skinned muscle fibres from rabbit psoas. Results were compared when tension was inhibited with either BeFx or 2,3-butanedione-monoxime (BDM) or modulated by altering myoplasmic [Ca2+]. With 3 mM total fluoride, 1 mM BeFx inhibited both tension and KS by approximately 50% (compared to 7-10 mM BDM and 50-100 microM A1F4-). Increasing [BeFx] to 10 mM further reduced tension to approximately 15% P0, but had little further effect on KS; with BDM and altered [Ca2+], KS scaled more proportionately with tension. Inhibition of tension and KS by BeFx was more rapidly reversible, compared with slow recovery from tension inhibition with A1F4- or Vi. Vu exhibited a complex dependence on [BeFx], being relatively unaffected by concentrations < or = 1 mM, and becoming inhibited steeply for [BeFx] above this level. With BDM, Vu co-varied more directly with force. Our results suggest that BeFx may induce a different cross-bridge state in fibres than do A1F4- or Vi, but all three analogues of Pi form complexes that mimic crossbridge states that follow ATP hydrolysis.

Calcium dependence of Donnan potentials in rigor: the effects of [Mg2+] and anions in isolated rabbit psoas muscle fibres

Contraction in vertebrate striated muscle is known to be dependent upon the binding of calcium ions to the regulatory protein troponin C (TnC). Our electrical (Donnan potential) studies of the subsarcomeric regions have revealed an electrical switching mechanism, which is sensitive to both cation concentration and to particular anions. In a buffer containing phosphate and chloride ions and at 2.7 mM Mg2+we observe a single charge transition at pCa506.8 in both A-and I-bands. At zero Mg2+the pCa50of the A-band transition is shifted to 8.0 and the I-band shows two transitions (pCa50~6.8 and ~8.2). Increasing [Mg2+] to 4.5 mM produces a complex effect between pCas 7 and 9 in both bands. All effects are abolished at 9 mM Mg2+.In a chloride-only buffer (imidazole) at zero Mg2+the direction of the charge transitions is reversed. In addition, two transitions (pCa50~8.5 and ~7.0) are evident in the A-band and three in the I-band (pCa50~8.5, ~7.4, ~6.7). In the presence of Mg2+, again the effects of pCa upon the Donnan potential are complex. In the A-band at 2.7 mM Mg2+two transitions of opposite sign predominate (pCa ~7 and ~8), whilst in the I band a single transition (pCa ~8.3) occurs in the same direction as that observed in phosphate buffer. At 4.5 mM Mg2+the ‘W’ shape observed in the corresponding phosphate buffer is preserved in both bands with similar pCa50s. This shape is also apparent in the 9 mM Mg2+solution.In these two buffer systems, the magnitude of the charge change in terms of electron binding is far larger than expected from simple Ca2+/Mg2+binding to troponin. In an acetate-only buffer, however, the Donnan potentials of the A-band and I-band were very similar in magnitude and the charge change across the full pCa curve is close to the expected value for Ca2+/Mg2+binding to troponinWe speculate that titin has a role in the calcium activation of striated muscle in vertebrates for four reasons. First, the effects of long-term storage of the glycerinated muscle; second, the action of [Mg2+] ions; third the effect of anions; and fourth, our published and unpublished observations of sarcomere-length dependence. We also demonstrate the validity of our methodology, relating the charge transitions that we observe to cation-binding studies of a more traditional nature.

Synthesis of a spin-labeled photoaffinity ATP analogue, and its use to specifically photolabel myosin cross-bridges in skeletal muscle fibers.

A spin-labeled photoaffinity ATP analogue 3'(2')-O-[4-[4-oxo-(4-amido-2,2,6,6-tetramethyl-piperidino-1-oxyl)]-benz oyl]benzoyl adenosine 5'-triphosphate (SL-Bz2ATP) was synthesized and used to photolabel myosin in muscle fibers. Previous work has shown that 3'(2')-O-(4-benzoyl)benzoyl adenosine 5'-triphosphate (Bz2ATP) photolabeled Ser-324 of the 50 kDa tryptic fragment of skeletal S1 heavy chain. In this work, [alpha-32P]SL-Bz2ATP was hydrolyzed and trapped as the diphosphate analogue with Co2+ and orthovanadate at the active site of myosin in rabbit psoas muscle fibers. After UV irradiation, the myosin heavy chain was the only protein band found to be significantly photolabeled as assayed by gel electrophoresis and radioactivity counting. The labeling was localized after brief trypsin digestion by SDS-PAGE to be on the 50 kDa tryptic fragment of the S1 heavy chain. Ca. 35% of the myosin in fibers was covalently photolabeled. The fibers photolabeled with SL-Bz2ATP had the same active tension and maximum shortening velocity as the control fibers. The resulting spin label on myosin was too mobile to report the orientation of the heads in fibers. Nonetheless, this is the first work to show the feasibility of utilizing active site binding and photoaffinity labeling to place covalent spectroscopic probes at the myosin active site in fibers with high specificity and yield without affecting mechanical function.

Effect of nucleotides on thermal transitions of myosin

The internal dynamics and the thermal stability of myosin in rabbit psoas muscle fibres in different intermediate states of the ATP hydrolysis cycle were studied by differential scanning calorimetry (DSC) and electron paramagnetic resonance (EPR) spectroscopy. Three overlapping endotherms were detected in rigor, in strongly binding and weakly binding state of myosin to actin. The transition at 58.4°C can be assigned to the nucleotide-binding domain. The transition at highest temperature represents the unfolding of the actin and the contributions arising from the actin-myosin interaction. The transition of 54°C reflects the interaction between the subunits of myosin. Nucleotide binding induced shifts of the melting temperatures and produced variations in the calorimetric enthalpy changes. The changes of the EPR parameters indicated local rearrangements of the internal structure in myosin heads.

Identification of Troponin C Antagonists from a Phage-displayed Random Peptide Library

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.

Image-analysis-based assessment of the effects of the "Ca2+-jump" technique on sarcomere uniformity.

A new image analysis-based technique was used to quantitatively examine the effects of the "Ca2+-jump" activation protocol on the maintenance of fiber quality in skinned rabbit psoas muscle fiber segments. Specifically, contractions in pCa 4.6 were preceded by short-duration "preactivation" soaks in a solution in which EGTA was replaced with the low-Ca2+ buffering capacity analog hexamethylenediamine-N, N, N', N'-tetraacetate, which facilitated rapid Ca2+ equilibration within the fiber segments. Fiber quality was assessed by examining the Fourier spectra of the muscle fiber images before, during, and after activation. Segment lengths were typically below 500 micrometer, thus allowing the majority of the sarcomeres to be visualized in the field of view (x200 and x400 magnification). The preactivation protocol resulted in less deterioration of fiber quality with repetitive activation. In addition, there was also a significant reduction in the time required to reach the 50% level of maximum tension, with no significant change in the maximum tension level.

Distributions of Calcium in A and I Bands of Skinned Vertebrate Muscle Fibers Stretched to Beyond Filament Overlap

Measurements were made of the distributions of total calcium along the length of A and I bands in skinned frog semitendinosus muscles using electron probe x-ray microanalysis. Since calcium in the water space was kept below the detection limit of the technique, the signal was assumed to reflect the distribution of calcium bound to myofilament proteins. Data from sarcomeres with overlap between thick and thin filaments showed enhancement of calcium in this region, as previously demonstrated in rabbit psoas muscle fibers in rigor (Cantino, M. E., T. S. Allen, and A. M. Gordon. 1993. Subsarcomeric distribution of calcium in demembranated fibers of rabbit psoas muscle. Biophys. J. 64:211–222). Such enhancement could arise from intrinsic non-uniformities in calcium binding to either thick or thin filaments or from enhancement of calcium binding to either filament by rigor cross-bridge attachment. To test for intrinsic variations in calcium binding, calcium distributions were determined in fibers stretched to beyond filament overlap. Calcium binding was found to be relatively uniform along both thick and thin filaments, and therefore cannot account for the increased calcium observed in the overlap region. From these results it can be concluded that the observed enhancement of calcium is due to an increase in calcium binding to myofilaments as a result of rigor attachment of cross-bridges to actin. The source of the enhancement is most likely an increase in calcium binding to troponin, although enhancement of calcium binding to myosin light chains cannot be ruled out.

X-ray diffraction studies on the structural changes of rigor muscles induced by binding of phosphate analogs in the presence of MgADP

To clarify the structure of the ATP hydrolysis intermediates (ADP.P i bound state) formed by actomyosin crossbridges, the effects of various phosphate analogs in the presence of MgADP on the structures of the thin and thick filaments in glycerinated rabbit psoas muscle fibers in the rigor state have been investigated by X-ray diffraction with a short exposure time using synchrotron radiation. When MgADP and phosphate analogs such as metallofluorides (BeF x=3,4 and AlF 4) and vanadate (VO 4(V i)) were added to rigor fibers in the presence of the ATP-depletion backup system, the intensities of the actin-based layer lines were markedly weakened. The greatest effect (∼50% decrease in intensity) was observed in the presence of BeF x among the analogs examined. The intensity distribution of the 5.9 nm actin-based layer line shifted towards that observed in the Ca 2+-activated fibers, while the first actin layer line at ∼1/36.7 nm −1 retained a rigor-like profile with an intensity weakened by ∼50%. The intensity of the equatorial 10 reflection increased while that of the 11 reflection changed little, resulting in only a small increase (∼1.7 fold) in the intensity ratio of the 10 to the 11 reflection. No resting-like pattern appeared upon the addition of MgADP and BeF x . These results indicate that a substantial fraction (∼40%) of the myosin heads dissociate from actin but the detached heads remain in the vicinity of the actin filaments when MgADP and BeF x bind. The states produced by binding phosphate analogs to a rigor muscle differ from the resting-like state produced by adding them to a contracting muscle (Takemori et al., J. Biochem. (Tokyo) 117 (1995) 603–608). Our conclusion put forward to explain the data is that one of the two heads of a crossbridge is detached and the other retains a rigor-like attachment.