HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fujita, S. Articles by Yamada, K. Search for Related Content PubMed PubMed Citation Articles by Fujita, S. Articles by Yamada, K.

Fluorescence changes of a label attached near the myosin active site on nucleotide binding in rat skeletal muscle fibres

Suguru Fujita, Tomoko Nawata and Kazuhiro Yamada

MS 8726 Received 7 September 1998; accepted after revision 11 December 1998.

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. Trinitrophenyl AMP (TNP-AMP) in the concentration range 10-300 µM induced an increase in fluorescence intensity at around 530 nm in skinned skeletal muscle fibres freshly obtained from rat psoas muscle.

2. The fluorescence intensity of the fibres depended on TNP-AMP concentration up to ~200 µM. The K_d of TNP-AMP binding to the muscle fibres was 38·0 ± 8·4 µM (mean ± s.d., n = 4 measurements) in three fibres. TNP-AMP fluorescence was readily washed out.

3. Various nucleotides affected the fluorescence of the fibres incubated in 20 µM TNP-AMP. MgATP (1 mM) and caged ATP (5 mM) reduced the fluorescence in 20 µM TNP-AMP by more than 40 % of the value measured in the absence of nucleotide.

4. When the fibres were stretched to almost no filament overlap, the extent of the quenching of the TNP-AMP (20 µM) fluorescence due to ATP binding was reduced by 14 %. This might be explained by assuming that the association of the thin filament affected the TNP-AMP fluorescence in muscle fibres.

5. The distance between the active site and the specific site for TNP was measured by the fluorescence resonance energy transfer between N-methylanthraniloyl-ATP (Mant-ATP) bound to the active site and the TNP-AMP bound to the TNP-specific site in muscle fibres. The results showed that the distance between the two may be less than 2 nm.

6. It may be concluded that the fluorescence intensity at 530 nm in skinned muscle fibres in low concentrations of TNP-AMP changes directly reflecting the conformational state of the nucleotide-binding region that is determined by the binding of nucleotides.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Myosins are molecular motors which generate force and movement by interacting with actin in the thin filament. The significance of myosins as molecular motors has been expanded by recent findings that they occur ubiquitously in a number of different forms in all eukaryotic cells where they drive such diverse processes as muscle contraction, cytokinesis and the transport of cellular organelles. Myosins contain at least one myosin head, or motor domain. Each myosin head contains binding sites for both ATP and actin, and utilizes the energy derived from ATP hydrolysis to produce force and movement when bound to actin. The highly organized sarcomere structure of interdigitating myosin (myosin II) and actin filaments in striated muscle has contributed much to the understanding of how ATP hydrolysis is related to the mechanism in which the myosin head produces force and motion on interaction with actin. It remains unclear, however, how the myosin head produces force and motion.

The molecular mechanism by which myosins transduce chemical energy into directed movement has been a subject of compelling interest for many years. Myosin and actomyosin ATPases have been intensively studied to characterize the intermediate species, and this has led to a detailed description of the hydrolysis of ATP, where conformational changes are associated with substrate binding and splitting and release of products (Trentham et al. 1976; Taylor, 1979). The changes in the intrinsic fluorescence of myosin have proven to be a sensitive indicator that is capable of defining the conformation of subfragment 1 (S1) of myosin (Weber et al. 1973; Bagshaw & Trentham, 1974). However, the nature of the conformational changes implied in the myosin ATPase mechanism is not well understood. The recently determined crystal structure of myosin-S1 (Rayment et al. 1993) should aid elucidation of the mechanism underlying molecular function. Structural studies of the myosin motor domain complexed with nucleotide analogues have suggested that the major conformational changes in the myosin head occur in association with a rearrangement of the domains coupled to a structural change in the COOH-terminal domain (Fisher et al. 1995).

Motor proteins are thus unique among enzymes. Associated with the hydrolysis of bound ATP, structural changes are induced within the nucleotide-binding region and propagated by way of the reactive thiol SH1-containing helix, or hinge region, which may be close to the putative force-generating motor, to the lever arm system (Holmes, 1997; Dominguez et al. 1998), and also to the actin-binding cleft (Rayment et al. 1993), leading to force production when calcium is present. Fluorescence spectroscopy may provide a useful means for following dynamic structural changes of the motor proteins due to its high sensitivity and sensitivity to the micro-environment. These structural changes occur in myosins integrated into filaments in muscle, but the properties of myosin heads when structurally integrated may be different from those in isolated proteins in solution. Suitable fluorescence probes in muscle fibres should reveal important information concerning the functionally significant changes in protein structure occurring in contracting muscle fibres.

Tanner et al. (1992) and Berger et al. (1996) labelled, in glycerol-extracted muscle fibres, one of the cysteine residues, Cys-707 (SH1), in the myosin heavy chain with iodoacetamidotetramethylrhodamine (IATR). The results showed that the orientation of the rhodamine probes reflected structural changes linked to nucleotide binding. However, Tao & Lamkin (1981) using fluorescence resonance energy transfer (FRET) measured the distance between the ATPase site and SH1, to which the N-(iodoacetyl)-N'-(5-sulpho-1-naphthyl)ethylenediamine (1,5-IAEDANS) moiety was attached. They concluded that despite the great influence that the two sites exert on each other it is unlikely that SH1 interacts directly with the ATPase site.

Here we report novel fluorescence signals that reflect structural changes of the nucleotide-binding region of myosin on binding nucleotides in muscle fibres. Trinitrophenyl (TNP) nucleotides (Fig. 1), fluorescent analogues of nucleotides, bind to a specific site that is located very close to the active site, as well as to the active site itself, of myosin heads in muscle fibres. The fluorescence arising from TNP-AMP, which binds only to the TNP-specific site (Tao & Lamkin, 1981), changes in intensity on binding of various nucleotides to the active site. Moreover, the fluorescence changes on nucleotide binding seem to be affected by the presence of actin. In a preliminary study, we have shown that the TNP-AMP fluorescence changes further to a higher level associated with force production in muscle fibres (Yamada et al. 1997).

		View larger version [in this window] [in a new window]
Figure 1. Chemical structure of TNP nucleotides The general structural formula for a TNP nucleotide, the ribose-modified chromophoric fluorescent analogue of ATP, ADP and AMP at neutral pH (Hiratsuka & Uchida, 1973). The anionic structure of the trinitrophenyl moiety is responsible for the fluorescence, pKa (-log of the acid dissociation constant) being 5·0 (Hiratsuka, 1976).

	METHODS

Top Abstract Introduction Methods Results Discussion References

Preparation of skinned muscle fibres

Fibres were obtained from the psoas muscle of Male Wistar rats. The animals ( > 12 weeks old) were anaesthetized with diethylether and pentobarbitone sodium (50-75 mg kg^-1), and killed by exsanguination. Bundles 3-4 mm in diameter and 40-50 mm in length were taken from the psoas muscle, then single fibres (diameter, 100 µm) were pulled out from the bundle in relaxing solution (for composition see Table 1). The fibres were treated with Triton X-100 (1 % w/v) in relaxing solution for 30-60 min to remove the cytoplasmic and internal membranes (Kurebayashi & Ogawa, 1991). All experiments were done within 12 h of excision of the bundles from the animal.

Chemicals and solutions

2' (or 3')-O-(2,4,6,-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) was obtained from Molecular Probes and purified on a Sephadex LH-20 column packed in water (Hiratsuka, 1976). N-Methylanthraniloyl-ATP (Mant-ATP) was synthesized by reaction of ATP with N-methylisatoic anhydride as described by Hiratsuka (1983). Purity of these substances was confirmed by thin layer chromatography (Hiratsuka, 1983).

The composition of experimental solutions is shown in Table 1. The concentrations of the solution constituents were calculated by solving the multiple binding equilibria of the components with stability constants given in Martell & Smith (1974). The concentration of Mg-ATP in the relaxing solution was 5·0 mM. Free Mg²⁺was 3·0 mM in all solutions. The ionic strength of all solutions was adjusted to 200 mM with potassium methanesulphonate (KCH₃SO₃). pH was adjusted to 7·0 with KOH. TNP-AMP (10-300 µM) and Mant-ATP (20 µM) were added to these solutions as required. Solutions for titrations with ATP (Fig. 7), for example, were prepared by mixing the rigor solution and 5·0 mM Mg-ATP relaxing solution, both of which contained 20 µM TNP-AMP. Resumptions of the equilibria among constituents were found to be negligibly small. ADP solutions were prepared following the same procedure. Adenosine-5'-O-(3-thiotriphosphate) (ATPS) and P³-(1-(2-nitrophenyl)ethyl)adenosine-5'-triphosphate (caged ATP) solutions were prepared by using these analogues in place of ATP. For FRET experiments, solutions were prepared by mixing rigor solution and 20 µM TNP-AMP rigor solution, both of which contained 20 µM Mant-ATP.

Table 1. Composition of solutions (mM)

The concentration of TNP-AMP was determined spectrophotometrically using an extinction coefficient of 18 500 M^-1 cm^-1 at 470 nm in 0·2 M Tris-HCl (pH 8·0). The value of the coefficient was taken to be the same as for TNP-ATP (Hiratsuka & Uchida, 1973) in view of the fact that the phosphate portion of TNP-adenine nucleotides has little effect on the chromophore at pH 8 (Hiratsuka, 1982). The concentration of Mant-ATP was determined from the extinction coefficient described by Hiratsuka (1983).

ATP, ADP and ATPS were obtained from Boehringer Mannheim. Methanesulphonic acid was obtained from Tokyo Kasei (Kogyo, Tokyo, Japan). Pipes, EGTA and caged ATP (highly purified lot, ATP < 0·008 %) were from Dojindo Laboratories (Kumamoto, Japan).

Fluorescence and mechanical measurements

The stainless steel trough assembly (Fig. 2) had three troughs, main (70 µl), central (5 µl) and front (100 µl). The front and bottom of the front trough had quartz windows. The trough assembly was fixed on a mechanical stage on a ball-bearing slide and could be lowered or made to slide fixed distances. The solutions in the main trough were removed by aspiration. The temperature of the trough assembly was maintained at 11-12°C by circulating thermostatic water. The sarcomere length was measured using a × 32 objective lens, a × 2·5 insertion lens and a × 10 eyepiece with graticule. The temperature of the silicone oil (KF96L, Shin-Etsu) in the front trough, in which muscle fibres were placed for fluorescence measurements, was controlled at 12-13°C.

		View larger version [in this window] [in a new window]
Figure 2. Schematic drawing of the apparatus for measurements of fluorescence, force and stiffness of single skinned muscle fibres Fibres were placed initially into relaxing solution in the main trough (left) of the trough assembly, the temperature of which was thermostatically controlled at 11-12 °C. After rigor was induced by removing ATP by exchanging relaxing for rigor solution in the main trough, the fibres were transferred to the central trough to incubate them in TNP-AMP in the absence or presence of nucleotides by lowering and sliding the trough assembly. For fluorescence measurements fibres were placed in the front oil trough (right) on top of the objective lens (× 20) of an inverted epi-fluorescence microscope. The front trough had quartz windows (QW) in the front and bottom and was filled with silicone oil maintained at a temperature of 12-13 °C. The specifications for the bandpass filters are for TNP-AMP fluorescence measurements.

In fluorescence experiments excitation was performed at 436 nm for TNP-AMP and at 365 nm for Mant (FRET experiments) by using strong lines of a super-quiet mercury-xenon lamp (L2481, Hamamatsu Photonics, Hamamatsu, Japan), attenuated to 12-25 % for TNP fluorescence and to 6 % for Mant fluorescence using neutral density filters. No appreciable photobleaching was detected within several tens of seconds. Fluorescence intensity was measured using an epifluorescence microscope (Axiovert 135 TV, Zeiss) with a long working distance (11 mm) × 20 objective lens and photo-multiplier tube (PMT) (R329-02, Hamamatsu) through dichroic mirrors and bandpass filters with a centre at 530 nm for TNP fluorescence and at 436 nm for Mant fluorescence. PMT was used in a steady state with a cathodal potential of -800 to -1000 V. The PMT output was amplified using a battery-operated current-input preamplifier (LI-76, NF Electronic Instruments, Yokohama, Japan) with a gain of 10⁸.

Isometric tension of muscle fibres was measured using a silicone beam strain gauge (AE 801, Akers, Horten, Norway), to which an electrolytically sharpened tungsten wire hook (shaft diameter, 100 µm) was fixed. The resonant frequency of the system was 1 kHz and the stiffness 2 mN µm^-1. Isolated skinned muscle fibres were tied to the hook with single silk filaments. The length of the fibres was 2 mm. For measuring stiffness the other end of the fibre was tied to a hook, fixed to a ceramic piezoelectric stack (P-841.30, Physik Instrumente GmbH, Waldbronn, Germany), which was driven by a current amplifier (E-865.10, Physik Instrumente GmbH). The command signal was a sinusoid (500 Hz) from a lock-in amplifier (Model 393, Ithaco, NY, USA), resulting in a sinusoidal change in fibre length of less than 0·1 %. The tension signals consisted of muscle fibre force and a superimposed sinusoidal component due to the length oscillation applied by the piezoelectric device. The in-phase and the quadrature stiffness was obtained as described by Goldman et al. (1984). Signals were recorded and stored on a Nicolet digital oscilloscope (12-bit, Model 440) at 10 kHz and were processed on a PC using Famos software (imc GmbH, Berlin, Germany). Signals were also recorded on a chart recorder (Linearcorder model WR 3320, Graphtec, Yokohama, Japan).

Experimental protocol

The muscle fibre (sarcomere length, 2·5 µm) was initially relaxed in 0·1 mM Mg-ATP relaxing solution in the main trough and then the solution was changed to rigor solution (Fig. 3). After tension and in-phase stiffness reached a steady state by further exchange of the solution the fibre was transferred to the front trough by lowering, sliding and raising again the trough assembly. The shutter of the fluorescence microscope was opened for a brief period (< 30 s) to record the background level of fluorescence. The fibre was then transferred to the central trough containing rigor solution with the required concentration of TNP-AMP. After equilibration for 10 min the fibre was transferred to the front trough and the fluorescence of the fibre was recorded. The fibre was returned to relaxing solution in the main trough. The same procedure was repeated several times for fluorescence measurements under different conditions in the same fibre.

		View larger version [in this window] [in a new window]
Figure 3. Chart records from fluorescence intensity measurements in single skinned muscle fibres This figure shows chart records from part of the fluorescence resonance energy transfer (FRET) measurements (see Methods). A shows, from top to bottom, fluorescence (Fl), in-phase stiffness (St) and force (F) in a fibre. The fibre was first equilibrated in a relaxing solution with reduced [ATP] (0·1 mM), then after several exchanges of solution (registered as spikes in the force record), the fibre was placed in the rigor solution (leftmost arrow). After the fibre produced steady force by one more exchange of the solution (see also stiffness record), the fibre was transferred to the front trough and the shutter in the light path was opened for a brief period to record the fluorescence signal. The fibre was transferred to the central trough filled with 20 µM Mant-ATP (centre arrow) for 10 min, then the fibre fluorescence was measured in the front trough. The fibre was transferred to the main trough filled with relaxing solution (rightmost arrow). In B the same fibre was incubated in 20 µM Mant-ATP plus 20 µM TNP-AMP for 10 min. Note that the fluorescence was reduced to about 70 % after subtracting the background signal (see Fig. 8). The time base (T) shown at the bottom shows ticks every 1 min and applies to A and B. A cathodal potential of -997 V was applied to the PMT. The stiffness is calibrated relative to maximum rigor stiffness (given a value of 1). Fibre diameter was ~75 µm, fibre length 2·2 mm and sarcomere length 2·5 µm. Trough temperatures: main, 11·4 °C; front, 12·5 °C.

FRET measurements

The efficiency (E) of FRET between probes was determined by measuring the fluorescence of the donor both in the presence (F_DA) and absence (F_D) of the acceptor as given by:

E = 1 - F_DA/F_D. (1)

The efficiency is related to the distance (R) between the donor and acceptor, and the Förster's critical distance (R₀), at which the transfer efficiency is equal to 50 % as follows:

E = R₀⁶/(R₀⁶ + R⁶). (2)

R₀ (in nm) can be derived from the following equation:

R₀⁶ = (8·79 × 10^-11)n^-4K² Q_DJ, (3)

where n is the refractive index of the medium, K² is the orientation factor, Q_D is the quantum yield of the donor in the absence of the acceptor, and J (defined by eqn (4)) is the spectral overlap integral between the donor emission (F_D()) and acceptor absorption (()) spectra.

J = ‚F_D() () ⁴ d/‚F_D() d. (4)

The details of the distance calculation based on FRET measurement have been described by Stryer (1978) and by Fairclough & Cantor (1978).

Data analyses

Parameters for fluorescence titrations with TNP-AMP, for the effects of nucleotides on fluorescence intensity and for FRET measurements were analysed by fitting equations using the Marquardt-Levenberg algorithm for least-squares estimation of parameters implemented in SigmaPlot 1.01 and 4.0 for Windows (Jandel Scientific, Sausalito, CA, USA). Statistical decision and estimation of significance were carried out by using Student's t test.

	RESULTS

Top Abstract Introduction Methods Results Discussion References

Fluorescence of muscle fibres in TNP-AMP

The fluorescence intensity of muscle fibres was increased in the presence of TNP nucleotides, and was readily reversed in the absence of TNP nucleotides. The steady-state fluorescence was increased by TNP-AMP to almost the same extent as by TNP-ADP (data not shown). This is in agreement with the findings of Moss & Trentham (1983) who showed that myosin S1 has three classes of sites for the TNP nucleotides, that the fluorescence increases little by binding of TNP-ADP to the active site, and that a secondary, TNP-specific, site is responsible for the major fluorescence enhancement observed on binding the TNP-nucleotides. Tao & Lamkin (1981) showed that TNP-AMP binds to the TNP-specific site possibly without interacting with the binding of nucleotides to the active site (see Discussion). Therefore, in order to simplify the system, we used TNP-AMP rather than TNP-ADP in the following experiments.

Figure 4 shows the effect of the duration of incubation in TNP-AMP on the fluorescence intensity of a fibre. The fluorescence intensity of the fibres in TNP-AMP became stable within 10 min (Fig. 4). Therefore the incubation time was set at 10 min. The data points were obtained by subtracting the background signal intensity, which was the signal arising from the microscope field and the fibres in rigor solution without TNP-AMP, from the intensity recorded in the presence of TNP-AMP.

		View larger version [in this window] [in a new window]
Figure 4. Fluorescence intensity of muscle fibres incubated in TNP-AMP Effects of the concentration of TNP-AMP, and the duration of incubation in TNP-AMP, on fluorescence intensity at 530 nm of a muscle fibre. Fluorescence intensity in the solution containing TNP-AMP became stable after about 10 min at all concentrations of TNP-AMP studied.

Figure 5 shows the fluorescence intensity of a fibre placed in silicone oil against TNP-AMP concentration. Fibres were equilibrated in the rigor solution containing various concentrations of TNP-AMP before transferring to oil so that the concentrations of TNP-AMP are for free TNP-AMP. The affinity of TNP-AMP for the muscle fibre, possibly for the myosin heads, may be obtained by curve fitting, taking into consideration that the free TNP-nucleotide also contributed to the fluorescence intensity. The continuous curve shows the relation obtained by fitting the following equation to the data points:

F = [L]/(Kd + [L]) + [L], (5)

where F is the fluorescence intensity in arbitrary units, [L] the free TNP-AMP concentration, Kd is the dissociation constant of TNP-AMP, and and are constants. The first and the second terms on the right hand side of this equation are related to the fluorescence intensity associated with the bound and the free analogue, respectively, and are shown in the figure by dashed lines. The fluorescence may be absorbed by TNP-AMP present (inner filter effect; Lakowicz, 1983), however, the effect is less than about 4 % at 540 nm and can be disregarded. The best fit was obtained at a K_d of 54·1 µM in the fibre in Fig. 5. The value of K_d was 38·0 ± 8·4 µM (mean ± 1 S.D.) in four titrations in three fibres. It should be noted here that at a concentration of 20 µM TNP-AMP, which is the concentration mostly used for labelling the fibres, the fluorescence intensity due to the free TNP-AMP present in the fibre was 21·6 ± 3·9 % (mean ± 1 S.D., n = 4). Because the fluorescence intensities due to bound and free TNP-AMP were evaluated separately, the factor of enhancement for fluorescence intensity of TNP-AMP due to binding to the fibre can be ascertained if the myosin head concentration in the fibre is known (150 µM, Ferenczi et al. 1984) or K_d is known. The enhancement factor was calculated to be 1·5 ± 0·6 (mean ± 1 S.D., n = 4).

		View larger version [in this window] [in a new window]
Figure 5. Fluorescence intensity of a muscle fibre and TNP-AMP concentration The continuous curve shows the result of fitting to eqn (5). The dashed curves show two components, one associated with TNP-AMP bound to the myosin heads, and the other with free TNP-AMP increasing linearly with TNP-AMP concentration. In this fibre the Kd of binding of TNP-AMP was estimated to be 54·1 µM.

Effects of nucleotides on TNP-AMP fluorescence

Fibres were equilibrated in the solutions containing TNP-AMP and nucleotides and then transferred to oil for fluorescence measurements. Figure 6 shows that the fluorescence intensity of the fibres in TNP-AMP (20 µM) was reduced by adding ATP. Tao & Lamkin (1981) have shown that binding of TNP-AMP is not affected by excess ATP in myosin S1. This may be taken to imply that the binding of the nucleotide to the active site will not perturb the binding of TNP-ATP (see Discussion). Therefore, the effect of ATP most probably reflects the conformational changes of the active site of the myosin heads induced by binding of the nucleotide. Figure 6 shows results of titrations (mean ± 1 S.D.) with MgATP (, n = 7), MgADP (, n = 7), MgATPS (, n = 4-7) and caged ATP (, n = 3).

		View larger version [in this window] [in a new window]
Figure 6. Effects of nucleotides on the TNP-AMP fluorescence of muscle fibres The fluorescence intensity of muscle fibres in TNP-AMP (20 µM) was affected by the addition of various nucleotides. Different symbols show results of titrations with ATP (), ADP (), ATPS (), and caged ATP (). The curve for ATP shows the result of fitting to eqn (6). Symbols and error bars show the means ± 1 S.D. (n = 7 for ATP and ADP, n = 4-7 (depending on the data points) for ATPS, and n = 3 for caged ATP).

The effect of the nucleotide binding to the active site was essentially a reduction in the intensity of the TNP-AMP fluorescence. The titration curve (Fig. 6) may reflect the affinity of each nucleotide for the active site. The curve drawn for ATP was obtained by fitting the following equation to the data points:

F = 1 - (1 - f_s)[N]/(K_d + [N]), (6)

where F is the relative fluorescence intensity normalized to that in 20 µM TNP-AMP without any nucleotide, [N] is the concentration of the nucleotide, K_d is the dissociation constant of the nucleotide binding to the active site, and f_s is the fractional relative fluorescence intensity when the active site is saturated with the nucleotide. K_d was found to be 60 µM and f_s 0·56. With 20 µM TNP-AMP the fluorescence due to free TNP-AMP contributes 21 % of the total fluorescence intensity (see above). On the other hand, the effect of excess nucleotides was a reduction in the fluorescence intensity by 56 % (Fig. 6). This is also compatible with the view that these nucleotides are not competing with TNP-AMP for the same site (see Discussion).

The protocol of the experiment was such that, while a steady state was established in the central trough, the observation was made in the front trough (see Methods). For the titration with ATP, in the time taken for the fluorescence measurements part of the ATP would have been hydrolysed to ADP. The fluorescence was measured before an appreciable rigor force developed, hence the exclusion of results from ATP concentrations of less than 200 µM. Therefore, the results should be taken as indicating that the K_d for ATP binding is less than 60 µM, which is the value derived from the curve fitting procedure described above.

During titrations with ADP, when incubating at concentrations of > 1 mM considerable force gradually developed over the 10 min period. This was probably caused by impurities and therefore the results for ADP concentrations of > 1 mM could not be taken as reliable and were omitted. The results of the titrations shown in Fig. 6 indicate that the K_d for ADP binding may be larger than has been reported previously under similar conditions (18 µM, Dantzig et al. 1991). This discrepancy could possibly be due to the presence of contaminants. Other possibilities could be the difference in temperature, or some ADP bound to the fibres (Thirlwell et al. 1994). Note that caged ATP and ATPS also affected the fluorescence in 20 µM TNP-AMP in a concentration-dependent manner (Fig. 6).

Effects of nucleotides on TNP-AMP fluorescence - effects of filament overlap

The results described above have indicated that various nucleotides affect the TNP-AMP fluorescence. In muscle fibres myosin heads also interact with actin in the thin filaments, and they are bound to actin when nucleotides are not present. Therefore it is of interest to see the effect of the presence of actin on the above-described fluorescence changes on nucleotide binding. This can be accomplished by changing the extent of the filament overlap in muscle fibres.

Fibres in 20 µM TNP-AMP were titrated with ATP first at near optimal length (sarcomere length, 2·5 µm) and then after the fibres had been stretched to almost no filament overlap (sarcomere length, 4·0 µm). In stretched fibres no force developed and stiffness remained unchanged when the fibre was deprived of ATP. Fluorescence of the stretched fibres in 20 µM TNP-AMP was reduced to 60 % possibly because the fibre mass within the optical field was reduced by the stretch. Figure 7 shows that changing the sarcomere length from optimal to almost no overlap reduced the fluorescence changes induced by ATP significantly by 14 %. These results perhaps indicate that the conformation of the nucleotide-binding site is affected by interacting with the thin filament.

		View larger version [in this window] [in a new window]
Figure 7. Effect of filament overlap on TNP-AMP fluorescence of muscle fibres in various concentrations of ATP Fibres in 20 µM TNP-AMP were titrated with ATP at near-optimal length (sarcomere length, 2·5 µm) (), and after being stretched to almost no overlap of filaments (sarcomere length, 4·0 µm) (). Mean ± 1 S.D. (n = 2-6). Curves show results of fitting to eqn (6). The effect of the stretch was significant at 0·2, 2 and 5 mM ATP: *P < 0·05, **P < 0·01.

Distance measurements between the active site and the TNP-specific site

The observations described above might be explained if TNP-AMP remains bound in the vicinity of the active site; the results would then reflect the structural changes occurring within the active site. Fluorescence resonance energy transfer (FRET) studies were therefore carried out to determine the distance between the active site and the TNP-specific site in the myosin head in muscle fibres. The fluorescent probe, Mant-ATP, was used as the donor attached at the active site, TNP-AMP being the acceptor bound at its specific site. Mant-ATP that was bound at the active site in muscle fibres should have been cleaved to Mant-ADP some time after the fibre was transferred to the silicone oil in the front trough. Figure 8 shows the results of measurements in six preparations (mean ± 1 S.D.). When the catalytic sites were labelled using 20 µM Mant-ATP (Ferenczi et al. 1989), TNP-AMP produced a marked quenching of the fluorescence of Mant-ADP, which was bound at the catalytic site (Fig. 8).

		View larger version [in this window] [in a new window]
Figure 8. Fluorescence resonance energy transfer (FRET) in muscle fibres Fluorescence of muscle fibres in 20 µM Mant-ATP was quenched by TNP-AMP. Fluorescence arising from Mant-ADP was monitored throughout. Continuous curve shows eqn (8) fitted to the data points. Mean ± 1 S.D. (n = 3-5). For details see text.

At 50 µM TNP-AMP, which was the highest concentration used in the present FRET study, the fluorescence emission of Mant-ATP was reduced by about 60 %. Part of this reduction might be due to the high optical density of TNP-AMP (Hiratsuka & Uchida, 1973; Moss & Trentham, 1983). However, the absorption of Mant-ATP fluorescence by TNP-AMP present in the fibre was estimated to be about 6 % (see Discussion). This arises because the diameter and accordingly the optical density of the muscle fibre is small. The fact that the fluorescence intensity was reduced by about 60 % at a TNP-AMP concentration of 50 µM indicated that this might be caused mostly by FRET. In the presence of excess ATP the fluorescence intensity in 20 µM Mant-ATP was reduced to 20 % (data not shown), which might be taken to be the fluorescence intensity due to the free Mant-ATP present within the fibre. That the fluorescence seemed to approach asymptotically to this value as the concentration of the TNP-AMP was increased might indicate that the efficiency of the FRET effect is high. In the Discussion the efficiency of the energy transfer is shown to be close to 99 %, and therefore the most probable distance between the active site and the TNP-AMP site is less than 2 nm.

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

Conformational changes of the active site of myosin heads on nucleotide binding in muscle fibres

The effect of nucleotide binding to the active site was essentially a reduction in the intensity of the TNP-AMP fluorescence. Because the location of the binding site for TNP-AMP is likely to be very close to the active site on the myosin heads, the fluorescence arising from TNP-AMP bound to the TNP-specific site may be directly affected by the conformational changes of the active site induced by binding of nucleotides. The fact that the fluorescence intensity of 20 µM TNP-AMP in excess ATP (0·6, see Results and Fig. 6) is much greater than that attributable to free TNP-AMP (0·21 in 20 µM TNP-AMP, see Results) is consistent with this. Moss & Trentham (1983) noted that TNP-adenosine fluorescence in S1 was reduced by ATP. As will be discussed below this was probably caused by a change in fluorescence of the protein-bound TNP-adenosine. We should point out here that Fig. 4 in Moss & Trentham (1983) was mislabelled so that 2 and 3 should be reversed in both frames. This has been confirmed with the authors.

From the fluorescence titration studies shown in Fig. 5, it can be deduced that TNP-AMP increases the fluorescence by a factor of 1·5 on binding to the muscle fibre, and at a concentration of 20 µM the fluorescence due to free nucleotide analogue contributes 21 % of the total fluorescence intensity (see Results). In excess ATP there are two possible explanations: either (1) the fluorescence of the protein-bound TNP-AMP changes and as a result the total fluorescence reduces to 56 % or (2) TNP-AMP dissociates and as a result the fluorescence reduces. As is further discussed below the first is the more likely to occur. If the second possibility were the case the observed fluorescence would have reduced to 21 % because the fibre was equilibrated to 20 µM TNP-AMP. If there were only partial TNP-AMP dissociation the changes in ATP would be less.

The changes in the intrinsic fluorescence of myosin have proven to be a sensitive probe for the detection of the conformational states (Morita, 1967; Weber et al. 1973; Bagshaw & Trentham, 1974; Johnson & Taylor, 1978), and have led to the development of the kinetic model for the contractile cycle (Trentham et al. 1976; Taylor, 1979). In skeletal muscle myosin one of the tryptophanyl residues, Trp-510, has been proposed as the residue responsible for the fluorescence changes (Hiratsuka, 1992). In muscle fibres intrinsic fluorescence measurements on myosin heads are difficult because of the UV range of the light for excitation. The fluorescence signal that has been reported here may be useful as an alternative for the dynamic detection of functionally significant structural properties of myosin heads in muscle fibres. In a preliminary study on flash photolysis of caged ATP in the TNP-AMP labelled fibres, we have detected a higher level of fluorescence intensity during maintained active contraction following photorelease of ATP at pCa of 4·5 than that in the absence of Ca²⁺ (Yamada et al. 1997).

We have detected different conformations of the nucleotide-binding region of the myosin head in muscle fibres associated with (1) thin filament-attached, nucleotide-free (rigor) and (2) detached, nucleotide (ATP)-bound and cleaved states. In excess ATP the crossbridges are populated mostly in a transient state of hydrolysis. It is not certain because of the presence of possible contaminants in ADP whether the thin filament-attached, nucleotide (ADP)-bound state can be separated (Fig. 6). These different states of the nucleotide-binding region of the crossbridges might be closely related to the states that occur during the crossbridge movement associated with muscle contraction (Holmes, 1997; Dominguez et al. 1998).

It is of interest that caged ATP (NPE-caged ATP) affects the TNP-AMP fluorescence. It is apparent that caged ATP binds to the active site with a K_d of about 2 mM (Fig. 6). It has been reported that caged ATP binds to myosin with a K_d in the region of 0·5-1 mM (Dantzig et al. 1989). Sleep et al. (1994) studied the inhibitory effect of caged ATP on the binding of MgATP to myofibrils, and have reported that caged ATP behaves as a simple competitive inhibitor of ATP binding with an inhibition constant of 1·6 mM. Thirlwell et al. (1995) also have reported that caged ATP compounds competitively inhibit unloaded, isotonic shortening of muscle fibres in the presence of Ca²⁺ with an inhibition constant of 1-2 mM. These fit very well with the present results with the TNP-AMP fluorescence. It may be that caged ATP binding to the active site of myosin does not significantly affect the binding of other nucleotides (Sleep et al. 1994). It is clearly of importance to realize, however, that caged ATP itself affects the fluorescence because caged ATP has been used in studying fluorescence transients in muscle fibres (Ferenczi et al. 1989; Yamada et al. 1997).

The affinity of ATPS for S1 is weaker than that of ATP and only 10 times that of ADP (Geeves, 1991; Resetar & Chalovich, 1995). However, particularly in the absence of Ca²⁺, the myosin nucleotide binding sites in myofibrils are saturated in 5 mM ATPS (Berger & Thomas, 1994). ATPS interacts with actomyosin in a manner similar to ATP, but is hydrolysed by a factor of 500 more slowly (Bagshaw et al. 1972). In ATPS the crossbridges would populate the actomyosin-ATPS and myosin-ATPS states (Dantzig et al. 1988; Gulick et al. 1997). On the other hand, in the presence of ATP the crossbridges are populated in a pre-steady state or transient state, i.e. myosin-ADP-P_i (Lymn & Taylor, 1971). Although comparison between the effects of ATP and ATPS on TNP-AMP fluorescence is of interest it is difficult to place much significance on the present results because of the lower affinity of ATPS for the active site and also because commercially available ATPS is reported to contain up to 10 % ADP.

It was shown in this study that actin affected the extent or nature of the structural changes which were induced by ATP binding to the active site of the myosin heads. This indicated that communication might exist between the nucleotide- and actin-binding sites. The critical components of the myosin molecule, the nucleotide- and the actin-binding site, are separated by at least 3·5 nm (Rayment et al. 1993). According to structural studies the narrow cleft that splits the central 50 kDa segment of the heavy chain is the communication route between the nucleotide- and actin-binding site.

Distance measurements - FRET studies

In the FRET experiments the fluorescence of Mant-ADP bound at the active site was measured at 436 nm. Therefore, it was critical to try to avoid the effect of relatively high absorption by TNP-nucleotides in the 400 to 500 nm range. When the concentration of free TNP-AMP was increased to 50 µM, which was the maximal concentration used in the present FRET measurements, the total concentration of TNP-AMP may have increased to as much as 200 µM, assuming the myosin head concentration within the fibre to be 150 µM (Ferenczi et al. 1984) and one to one stoichiometry for TNP-AMP binding. An optical density (D) of 0·03 was calculated from the molar extinction coefficient of TNP-AMP multiplied by the TNP-AMP concentration in the fibre (200 µM) and by the length of the light path (100 µm). The molar extinction coefficient of TNP-AMP at 436 nm was taken to be 18 500 M^-1 cm^-1 (see Methods and Fig. 2 in Hiratsuka & Uchida, 1973). From the relation D = log(100/T), the percentage transmission (T) is 94 %. This indicates that the Mant fluorescence may be quenched by only 6 % due to absorption by TNP-AMP.

The extent of reduction of the Mant-ATP fluorescence by TNP-AMP may depend on two factors, absorption by TNP-AMP and by FRET, as described above.

F = 1 - (1 - f_m)E - f_m(1 - 10^-D)

- (1 - f_m - (1 - f_m)E ) (1 - 10^-D), (7)

where F is the observed relative fluorescence intensity normalized to that in zero TNP-AMP concentration, f_m the fraction of the observed fluorescence due to the free Mant-ATP, E the efficiency of the energy transfer, the fraction of TNP-AMP binding sites of the myosin head which is occupied by TNP-AMP ([TNP-AMP]/(K_d + [TNP-AMP])), and D the optical density due to both free and bound TNP-AMP of the fibre. By rearranging eqn (7) we obtain:

F = (1 - (1 - f_m)E) × 10^{-d(1 + [M] /[TNP-AMP])}, (8)

where d is the optical density of the fibre due to free TNP-AMP, [M] the myosin head concentration of the muscle fibre (150 µM) and [TNP-AMP] the TNP-AMP concentration. Equation (8) was fitted to the observed values, making E and K_d variables and fixing f_m at 0·2, which is the relative Mant-ATP fluorescence in excess ATP (see above). The continuous curve in Fig. 8 shows the results of a best fit. The efficiency of energy transfer was found to be 99·4 % with a standard error of the estimate of 9 % according to the algorithm used for non-linear curve fitting (see Methods). K_d was 25 µM with a standard error of 5 µM and coincided well with the value obtained by fluorescence titration with TNP-AMP (38 µM, see Results).

J was calculated to be 0·79 × 10¹⁵ M^-1 cm^-1 nm⁴. By taking n = 1·4, K² = 2/3 and Q_D = 0·35 (Hiratsuka, 1984), Förster's critical distance (R₀) was calculated to be 4·02 nm. Unfortunately, Mant- and TNP-nucleotide as the donor-acceptor pair are not well suited to the exact determination of the short distance of less than 2 nm in FRET because the Förster's critical distance (R₀, 4 nm) is more than twice as large (Fairclough & Cantor, 1978). Note that in eqn (2) as E becomes more than 90 %, the distance (R) becomes less than 70 % (2·8 nm) of R₀, and as E becomes more than 98·5 %, R becomes less than 50 % (2 nm) of R₀ (see Fig. 3 in Fairclough & Cantor, 1978). The above results may be taken to imply that the TNP-specific site is located less than 2 nm from the active site, and therefore the structure of the TNP-specific site may be affected by the binding and possibly by the subsequent cleavage of the nucleotides at the active site. Tryptophanyl residues including Trp-131 could be involved possibly by a stacking process for the binding of TNP nucleotides to the specific site other than the active site.

We have shown that the TNP-specific site may be located within 2 nm of the active site. Moss & Trentham (1983) showed in FRET studies that the distance between the active site and the essential light chain (ELC) of S1 is 5·7 nm, and also that the TNP-specific site is located 4 nm from ELC. From these results the distance between the active site and the TNP site can be extrapolated as 1·7 nm if these sites are aligned with the labelled site of ELC. Our results show the distance between the active site and the TNP site to be 2 nm which seems to indicate that all these sites are aligned.

Ribose-modified ATP analogues

A ribose-modified chromophoric fluorescent analogue of ATP, TNP-ATP, was synthesized by Hiratsuka & Uchida (1973). This analogue is suited to physiological studies in muscle fibres because it has absorption maxima at visible wavelengths at neutral pH. This analogue is hydrolysed by heavy meromyosin (HMM) in the presence of magnesium (Hiratsuka et al. 1977). Changes in the absorption characteristics suggested that not only the phosphate portion but also the chromophoric portion of the analogue may bind to HMM (Hiratsuka & Uchida, 1973). The K_d of TNP-ADP binding to HMM was shown to be 70 µM from absorption characteristics (Hiratsuka & Uchida, 1973), while it was shown to be 0·8 µM from fluorescence (Hiratsuka, 1976). In both of these studies the number of binding sites per HMM molecule was 2.

Moss & Trentham (1983) studied the binding of TNP-ADP to S1 from skeletal muscle myosin. The changes in the intrinsic protein fluorescence arising from tryptophanyl residues within the active site indicated one binding site with a K_d of 0·3 µM, while binding of TNP-ADP to this site was associated with very little change in fluorescence. In addition, Moss & Trentham (1983) showed a second binding site for TNP-ADP, which was weaker in affinity (K_d > 30 µM) than at the active site but which when populated greatly enhanced the TNP-ADP fluorescence. Tao & Lamkin (1981) showed, from FRET studies with TNP-nucleotides and S1, in which SH1 was labelled with 1,5-IAEDANS (AEDANS-S1), that TNP-ADP binds both to the TNP-specific site and to the active site of S1, while TNP-AMP binds exclusively to the TNP-specific site.

According to Tao & Lamkin (1981) TNP-AMP quenches the AEDANS-S1 fluorescence to a certain extent, which was little affected by excess ATP. Moreover, ATP only partially reversed the quenching caused by either TNP-ADP or TNP-AMP. These results clearly indicated that some quenching occurred by energy transfer to TNP-nucleotides bound at a site other than the active site. Fluorescence lifetime measurements by these authors indicated that under conditions where the TNP-ADP to AEDANS-S1 ratio exceeded 1·0 in the presence of excess ATP a two component decay was observed, corresponding to a species with neither site occupied by TNP-ADP and a species with only the TNP-specific site occupied by TNP-ADP. This most probably indicated that a single class of TNP-specific site was involved. From this we conclude that TNP-AMP binds only to the TNP-specific site and not to the active site, while TNP-ATP and TNP-ADP bind to both the TNP-specific site and the active site of myosin in muscle fibres (see Results).

TNP-nucleotides are known also to react with the cysteine residues of S1, possibly the SH1 sulfhydryl group (Moss & Trentham, 1983). The rate of reaction is slow, however, and the second-order rate constant is 0·6 M^-1 s^-1 at pH 8 and 23°C. Assuming this rate also applies to the present experimental conditions, a simple calculation shows that the proportion of SH1 in the myosin head that has reacted with TNP nucleotides during the incubation period of 10 min does not exceed 1 %.

Throughout this work it has been apparent that fluorescence studies on muscle fibres have a number of advantages over those on solutions. (1) The fibre is an open system in which substances diffuse into the fibre freely; however, it can also be made a closed system by placing it in oil, (2) due to the small diameter of the fibres the inner filter effect (Lakowicz, 1983) is much less pronounced than in the conventional cuvette for fluorescence spectroscopy, (3) the concentration of myosin, as well as that of actin, is high, and (4) by changing the the filament overlap of the fibres the interaction between myosin and actin can be controlled quantitatively and reversibly. The present study has also shown that FRET studies are feasible in muscle fibres and possibly in other cells using fluorescence microscopes.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Bagshaw, C. R., Eccleston, J. F., Trentham, D. R., Yates, D. W. & Goody, R. S. (1973). Transient kinetic studies of the Mg2+-dependent ATPase of myosin and its proteolytic subfragments. Cold Spring Harbor Symposium on Quantitative Biology 37, 127-135.
Bagshaw, C. R. & Trentham, D. R. (1974). The characterization of myosin-product complexes and of product-release steps during the magnesium ion-dependent adenosine triphosphatase reaction. Biochemical Journal 141, 331-349	[Medline]
Berger, C. L., Craik, J. S., Trentham, D. R., Corrie, J. E. T. & Goldman, Y. E. (1996). Fluorescence polarization of skeletal muscle fibres labeled with rhodamine isomers on the myosin heavy chain. Biophysical Journal 71, 3330-3343	[Abstract]
Berger, C. L. & Thomas, D. D. (1994). Rotational dynamics of actin-bound intermediates of the myosin adenosine triphosphatase cycle in myofibrils. Biophysical Journal 67, 250-261	[Abstract]
Dantzig, J. A., Goldman, Y. E., Luttmann, M. L., Trentham, D. R. & Woodward, S. K. A. (1989). Binding of caged ATP diastereoisomers to rigor cross-bridges in glycerol-extracted fibres of rabbit psoas muscle. The Journal of Physiology 418, 61P.
Dantzig, J. A., Hibberd, M. G., Trentham, D. R. & Goldman, Y. E. (1991). Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. The Journal of Physiology 432, 639-680	[Abstract]
Dantzig, J. A., Walker, J. W., Trentham, D. R. & Goldman, Y. E. (1988). Relaxation of muscle fibres with adenosine 5'-[-thio]triphosphate (ATP[S]) and by laser photolysis of caged ATP[S]: Evidence for Ca2+-dependent affinity of rapidly detaching zero-force cross-bridges. Proceedings of the National Academy of Sciences of the USA 85, 6716-6720	[Medline]
Dominguez, R., Freyzon, Y., Trybus, K. M. & Cohen, C. (1998). Crystal structure of a vertebrate smooth muscle myosin motor domain and its complex with the essential light chain: visualization of the pre-power stroke state. Cell 94, 559-571	[Medline]
Fairclough, R. H. & Cantor, C. R. (1978). The use of singlet-singlet energy transfer to study macromolecular assemblies. Methods in Enzymology 28, 347-379.
Ferenczi, M. A., Homsher, E. & Trentham, D. R. (1984). The kinetics of magnesium adenosine triphosphate cleavage in skinned muscle fibres of the rabbit. The Journal of Physiology 352, 575-599	[Abstract]
Ferenczi, M. A., Woodward, S. K. A. & Eccleston, J. F. (1989). The rate of mant-ADP release from cross-bridges of single skeletal muscle fibres. Biophysical Journal 55, 441.
Fisher, J. F., Smith, C. A., Thoden, J. B., Smith, R., Sutoh, K., Holden, H. M. & Rayment, I. (1995). X-ray structure of the myosin motor domain of Dictyostelium discoideum complexed with MgADP·BeFx and MgADP·AlF4-. Biochemistry 34, 8960-8972.
Geeves, M. A. (1991). The dynamics of actin and myosin association and the crossbridge model of muscle contraction. Biochemical Journal 274, 1-14	[Medline]
Goldman, Y. E., Hibberd, M. G. & Trentham, D. R. (1984). Relaxation of rabbit psoas muscle fibres from rigor by photochemical generation of adenosine-5'-triphosphate. The Journal of Physiology 354, 577-604	[Abstract]
Gulick, A. M., Bauer, C. B., Thoden, J. B. & Rayment, I. (1997). X-ray structures of the MgADP, MgATPS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain. Biochemistry 36, 11619-11628	[Medline]
Hiratsuka, T. (1976). Fluorescence properties of 2'(or 3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate and its use in the study of binding to heavy meromyosin ATPase. Biochimica et Biophysica Acta 453, 293-297	[Medline]
Hiratsuka, T. (1982). Biological activities and spectroscopic properties of chromophoric and fluorescent analogs of adenosine nucleoside and nucleotides, 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) adenosine derivatives. Biochimica et Biophysica Acta 719, 509-517	[Medline]
Hiratsuka, T. (1983). New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochimica et Biophysica Acta 742, 496-508	[Medline]
Hiratsuka, T. (1984). Affinity labeling of the myosin ATPase with ribose-modified fluorescent nucleotides and vanadate. Journal of Biochemistry 96, 147-154	[Medline]
Hiratsuka, T. (1992). Spatial proximity of ATP-sensitive tryptophanyl residue(s) and Cys-697 in myosin ATPase. Journal of Biological Chemistry 267, 14949-14954	[Abstract]
Hiratsuka, T. & Uchida, K. (1973). Preparation and properties of 2'(or 3')-O-(2,4,6-trinitro-phenyl) adenosine 5'-triphosphate, an analog of adenosine triphosphate. Biochimica et Biophysica Acta 320, 635-647	[Medline]
Hiratsuka, T., Uchida, K. & Yagi, K. (1977). Heterogeneous reaction of heavy meromyosin ATPase with 2'(or 3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate. Journal of Biochemistry 82, 575-583	[Medline]
Holmes, K. C. (1997). The swinging lever-arm hypothesis of muscle contraction. Current Biology 7, R112-118	[Medline]
Johnson, K. A. & Taylor, E. W. (1978). Intermediate states of subfragment 1 and actosubfragment 1 ATPase: reevaluation of the mechanism. Biochemistry 17, 3432-3442	[Medline]
Kurebayashi, N. & Ogawa, Y. (1991). Discrimination of Ca2+-ATPase activity of the sarcoplasmic reticulum from actomyosin-type ATPase activity of myofibrils in skinned mammalian skeletal fibres: distinct effects of cyclopiazonic acid on the two ATPase activities. Journal of Muscle Research and Cell Motility 12, 355-365	[Medline]
Lakowicz, J. R. (1983). Principles of Fluorescence Spectroscopy. Plenum Press, New York.
Lymn, R. W. & Taylor, E. W. (1971). Mechanism of adenosine triphosphate hydrolysis of actomyosin. Biochemistry 10, 4617-4624	[Medline]
Martell, A. E. & Smith, R. M. (1974). Critical Stability Constants, vol. 1-6. Plenum Press, New York.
Morita, F. (1967). Interaction of heavy meromyosin with substrate. I. Difference in ultraviolet absorption spectrum between heavy meromyosin and its Michaelis-Menten complex. Journal of Biological Chemistry 242, 4501-4506	[Medline]
Moss, D. J. & Trentham, D. R. (1983). Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle. Biochemistry 22, 5261-5270	[Medline]
Rayment, I., Rypniewski, W. R., Schmidt-Base, K., Smith, R., Tomchick, D. R., Benning, M. M., Winkelmann, D. A., Wesenberg, G. & Holden, H. M. (1993). Three-dimensional structure of myosin subfragment-1: a molecular motor. Science 261, 50-58	[Medline]
Resetar, A. M. & Chalovich, J. M. (1995). Adenosine 5'-(-thiotriphosphate): an ATP analog that should be used with caution in muscle contraction studies. Biochemistry 34, 16039-16045	[Medline]
Sleep, J., Herrmann, C., Barmann, C. & Travers, F. (1994). Inhibition of ATP binding to myofibrils and acto-myosin subfragment 1 by caged ATP. Biochemistry 33, 6038-6042	[Medline]
Stryer, L. (1978). Fluorescence energy transfer as a spectroscopic ruler. Annual Review of Biochemistry 47, 819-846	[Medline]
Tanner, J. W., Thomas, D. D. & Goldman, Y. E. (1992). Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres. Journal of Molecular Biology 223, 185-203	[Medline]
Tao, T. & Lamkin, M. (1981). Excitation energy transfer studies on the proximity between SH1 and the adenosinetriphosphatase site in myosin subfragment 1. Biochemistry 20, 5051-5055	[Medline]
Taylor, E. W. (1979). Mechanism of actomyosin ATPase and the problem of muscle contraction. CRC Critical Reviews in Biochemistry 6, 103-164	[Medline]
Thirlwell, H., Sleep, J. A. & Ferenczi, M. A. (1995). Inhibition of unloaded shortening velocity in permeabilized muscle fibres by caged ATP compounds. Journal of Muscle Research and Cell Motility 16, 131-137	[Medline]
Trentham, D. R., Eccleston, J. F. & Bagshaw, C. R. (1976). Kinetic analysis of ATPase mechanisms. Quarterly Reviews of Biophysics 9, 217-281	[Medline]
Weber, M. M., Szent-Györgyi, A. G. & Fasman, G. D. (1973). Fluorescence studies on the interaction between heavy meromyosin and magnesium ions. Effect of EDTA. Journal of Mechanochemistry & Cell Motility 2, 35-43.	[Medline]
Yamada, K., Fujita, S. & Nawata, T. (1997). Fluorescence changes of a label attached near the myosin active site on nucleotide binding and force generation in skeletal muscle fibres. XXXIII Congress of the International Union of Physiological Sciences P034.01.

Acknowledgements

We thank Drs D. R. Trentham, T. Hiratsuka, T. Arata, K. Horiuti and K. Kagawa for advice. This work was supported in part by Grant-in-Aid for Scientific Research (62480108 and 05454683) from the Ministry of Education, Science and Culture and a grant from the Ciba Geigy Foundation (Japan) for the Promotion of Science.

Corresponding author

K. Yamada: Department of Physiology, Oita Medical University, Oita 879-5593, Japan.

Email: yamadakz{at}oita-med.ac.jp

This Article Abstract Full Text (PDF) Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Fujita, S. Articles by Yamada, K. Search for Related Content PubMed PubMed Citation Articles by Fujita, S. Articles by Yamada, K.