Differing ADP release rates from myosin heavy chain isoforms define the shortening velocity of skeletal muscle fibers.

To understand mammalian skeletal myosin isoform diversity, pure myosin isoforms of the four major skeletal muscle myosin types (myosin heavy chains I, IIA, IIX, and IIB) were extracted from single rat muscle fibers. The extracted myosin (1-2 microg/15-mm length) was sufficient to define the actomyosin dissociation reaction in flash photolysis using caged-ATP (Weiss, S., Chizhov, I., and Geeves, M. A. (2000) J. Muscle Res. Cell Motil. 21, 423-432). The ADP inhibition of the dissociation reaction was also studied to give the ADP affinity for actomyosin (K(AD)). The apparent second order rate constant of actomyosin dissociation gets faster (K(1)k(+2) = 0.17 -0.26 microm(-1) x s(-1)), whereas the affinity for ADP is weakened (250-930 microm) in the isoform order I, IIA, IIX, IIB. Both sets of values correlate well with the measured maximum shortening velocity (V(0)) of the parent fibers. If the value of K(AD) is controlled largely by the rate constant of ADP release (k(-AD)), then the estimated value of k(-AD) is sufficiently low to limit V(0). In contrast, [ATP]K(1)k(+2) at a physiological concentration of 5 mm ATP would be 2.5-6 times faster than k(-AD).

Muscle contraction results from a cyclical interaction of myosin cross-bridges with actin driven by ATP hydrolysis. Skeletal muscle fibers show characteristic mechanical properties including shortening velocity, power output (1)(2)(3)(4), and ATPase activity (3,5). It is now clear that all of these properties are to a large extent determined by the isoforms of the myosin heavy chains expressed in individual muscle cells (6,7). Biochemical and structure-function studies have attempted to define the underlying molecular basis of these differing mechanochemical properties of myosins within the skeletal muscle myosins and in the wider family of myosins.
It is generally accepted that the sequence of events in the actomyosin cross-bridge cycle is essentially the same for all muscle myosins. The different properties can therefore be attributed to modulation of the rates and equilibrium constants (and hence free energy changes) of individual molecular events by changes in myosin sequence. Early studies suggest a corre-lation between the maximum shortening velocity of a muscle fiber (i.e. the velocity in the absence of any mechanical load) and the overall ATPase rate (8) for a range of muscle types, since both reflect the underlying speed of the cross-bridge cycle.
Detailed biochemical kinetic studies of myosin isoforms were pioneered in 1980 by Marston and Taylor (9) using four different muscle types of the chicken. They found that the relative rates of most of the steps in the ATPase cycle varied with myosin isoform but that most events remained too fast to contribute to defining the overall rate of ATP hydrolysis and, thus, the overall rate of energy transduction. For example, the rate of the ATP cleavage step was too fast to limit the overall cycling rate. They proposed that the rate-limiting step in the ATPase cycle was associated with the overall rate of reattachment of actin to the myosin products complex (M⅐ADP⅐P i ) and the product (P i and ADP) release. In a paper of great insight, Siemankowski et al. (10) estimate the rate of release of ADP from the actomyosin complex for a series of myosins isolated from a wide variety of sources including different muscle types (smooth, cardiac, and skeletal muscle) and different species (chicken, rat, rabbit). This work proposed that the rate of ADP release from a myosin was a major contributor to defining the maximum shortening velocity of a muscle expressing that myosin.
Recently the focus of attention has largely switched to studies of myosins of defined sequence and 3-dimensional structure. Such studies have examined naturally occurring variants of myosin to look for structure-function relationships. The overexpression of modified versions of myosin have allowed the investigation of chimeric myosins and myosins carrying specific mutations (e.g. cardiac myopathy point mutations) to pinpoint the parts of myosin responsible for particular properties (11)(12)(13)(14).
In a series of recent studies the mechanochemical properties of single muscle fibers and the myosins isolated from such fibers were compared (3,4,15,16). In particular, the work showed that significant changes in shortening velocity have been observed in muscle fibers expressing myosin isoforms that have few differences in sequence. For example the ␤-isoform of rat and human myosin heavy chain (␤-MHC 1 or MHC-I) are Ͼ95% identical in the myosin motor domain, with only 14 nonconservative point mutations throughout the 860-amino acid sequence, yet differ in motility properties; the myosins differ by a factor of 2 in the in vitro motility assays, and muscle fibers expressing the myosin have a 2-fold difference in maximum shortening velocity (16).
We therefore considered that a study of the biochemical kinetic properties of the mammalian myosin isoforms may prove of interest in testing the hypothesis of Siemankowski et al. (10) on a series of closely related myosins whose contractile and energetic properties are well characterized. Although there have been many studies of the mechanical properties of mammalian muscle fibers (both intact and skinned), biochemical characterization of mammalian-striated MHC isoforms has been limited because of the difficulty of isolating pure heavy chain isoforms. There is no effective overexpression system for mammalian-striated muscle myosin isoforms. Isolation of myosin from bulk muscle (as used by Siemankowski and White (17)) results in a preparation containing mixed isoforms, since most mammalian muscles contain multiple myosin isoforms. In contrast, single isolated muscle fibers often contain a single MHC isoform (18). We recently demonstrated that the amount of myosin that can be isolated from a 2-cm-long single muscle fiber of rat is sufficient to characterize the ATP-induced dissociation reaction and the affinity of ADP for actomyosin using a flash photolysis apparatus developed for this purpose (19). We report here the first kinetic characterization of all four MHC skeletal isoforms (type I, IIA, IIB, and IIX) expressed in adult rat muscle and show that both the rate of ATP induced dissociation of actomyosin and the rate of release of ADP from actomyosin becomes faster as the maximum shortening velocity of muscle fibers expressing the MHC isoform increases.

MATERIALS AND METHODS
Preparation of Proteins-The myosin used in these measurements was extracted from 15-mm-long single muscle fibers dissected from bundles of fresh soleus and psoas rat muscle. The fibers were manually dissected, chemically skinned (according to Bottinelli et al. (4)), and cut in two segments. The smaller segment (ϳ2 mm) was dissolved in 20 l of standard buffer solution (20) and used for MHC isoform identification by SDS-polyacrylamide gel electrophoresis on 8% polyacrylamide gels prepared according to Talmadge and Roy (21). Electrophoresis was run for 24 h at 250 V. Gels were silver-stained, and in the region of the myosin heavy chain (molecular mass, ϳ220 kDa) four bands were separated corresponding to the four MHC isoforms. Fig. 1A shows a gel with one example each of a fiber expressing a single MHC together with a sample containing a mixture of all four isoforms.
The major part of the fiber (ϳ13 mm) was incubated in 30 l of myosin extraction buffer (100 mM KH 2 PO 4 , 50 mM K 2 HPO 4 , 0.3 M KCl) for 3 h on ice, after which the fiber was removed and discarded. The myosin solution was dialyzed twice in a microdialysis system developed with Prof. J. Sparrow (University of York) against 0.5 ml of experimental buffer containing 500 mM KCl, 20 mM MOPS, and 5 mM MgCl 2 at pH 7.0 and used without further purification.
Rabbit actin was purified by the method of Spudich and Watt (22). Its molar concentration was determined by absorbance at 280 nm (⑀ 1% ϭ 1.104 cm Ϫ1 ) and a molecular mass of 42 kDa (23). Phalloidin-stabilized F-actin was used in all experiments and was made by incubating 10 M F-actin and 10 M phalloidin in experimental buffer overnight (24).
Flash Photolysis Apparatus-The flash photolysis system was constructed in Canterbury and has been described in detail by Weiss et al. (2000) (19). The quartz sample cuvette (1.5 ϫ 1.5 ϫ 20 mm, standard sample volume of 20 l) in the core of the optical system can be exposed to a 5-ns flash from a Nd-YAG laser (Surelite I-10, Continuum) with a pulse energy of up to 70 mJ (and a maximum of 15 mJ used in these experiments) at a wavelength of 355 nm. This provides light for the release of ATP from the cATP and was used to liberate up to 45 M ATP from 0.5-1 mM cATP at a rate of 90 s Ϫ1 . The optical bench was designed for simultaneous time-resolved detection of transmission changes and light-scattering changes. For detection a 100-W halogen lamp (Xenophot HLX 12 V, Osram) was used. The sample was exposed to white light of wavelengths of Ͼ389 nm (KV 389 filter, Schott, Germany) to prevent photolysis of cATP by light of a shorter wavelength). The speed and extent of ATP release from cATP in each transient were estimated via transmission changes at 405 nm, which monitors the photochemical breakdown of cATP (19). Light scattering at 90°to the incident light was used to monitor the dissociation of actomyosin. 50,000 data points were collected per transient then compressed to 560 data points on a quasi-logarithmic time scale for computer analysis using Microcal Origin 6.0 Professional (26).
All flash photolysis experiments with myosin were performed at 22°C in experimental buffer containing 10 mM fresh DTT to eliminate photolysis breakdown products. To reuse the same sample in multiple flash experiments, the sample contained sufficient additional enzymes to break down either ATP (0.03 units/l hexakinase, 1 mM glucose, and 100 M Ap5A) or ATP and ADP (0.01 units/l apyrase) on a minute time scale.
Statistical Analysis-Data were expressed as mean Ϯ S.D. Statistical significance of the differences between the means was assessed by variance analysis followed by the Student-Newman-Keuls test. A probability of less than 5% was considered significant.
Schemes and Equations-The kinetic model of actomyosin dissociation by ATP and its inhibition by ADP is defined by Scheme I, and the kinetic equation used to analyze the reaction is shown in Equation 1, where A, M, T, and D are actin, myosin heads, ATP, and ADP respectively. K 1 (ϭ k ϩ1 /k Ϫ1 ) is the association constant for formation of the A⅐M⅐T complex, and k ϩ2 is the rate constant for the isomerization of A⅐M⅐T to A-M⅐T; the subsequent dissociation of actin from this complex is assumed to be very fast. K AD (ϭk ϪAD /k ϩAD ) defines the dissociation constant for ADP binding to A⅐M (17).
The relationship between the maximal shortening velocity of a muscle fiber (V 0 ) and the minimal value of the rate constant (k min ) for the molecular event that limits the velocity was defined by Siemankowski et al. (10) and is shown in Equation 2 in which S L represents the half sarcomere length, and d represents the maximum allowed axial crossbridge translation.

RESULTS
Single fibers of ϳ15 mm in length were dissected from fresh preparations of soleus and psoas rat muscles. A small part of the fiber (ϳ2 mm) was used to identify the content of myosin as described under "Materials and Methods" (see Fig. 1A); the remaining part was used to isolate pure myosin isoforms. Single soleus fibers yielded myosin preparations of pure isoforms of MHC-I (38%), MHC-IIA (32%), and preparations containing mixed isoforms (30%). For psoas fibers, the preparations gave isolated isoforms of 47% pure MHC-IIB and 49% pure MHC-IIX. Mixed isoforms were found in 4% of the psoas fibers. Muscle fibers that did not contain a pure isoform were discarded, and pure myosin isoforms were extracted from the remaining fibers into a final volume of less than 50 l using micro incubation and dialysis methods. Comparison of the staining intensities of the gel in Fig. 1B shows that the extraction method provides yields of ϳ2 g of myosin with little contamination with actin or other proteins. The figure also suggests that more than 2/3 of the total myosin content of the fiber was extracted using this method. Thus, it is unlikely that any major improvements can be made in the quantity of myosin extracted.
ATP Induced Dissociation of Actomyosin-The binding of ATP to actomyosin causes fast and irreversible dissociation of the complex, and after all the ATP is hydrolyzed, the complex reforms. The rate of hydrolysis of ATP by actomyosin under these conditions is relatively slow (Ͼ10 min for 45 M ATP under the conditions used), and to allow a faster elimination of ATP and ADP, apyrase was added to the samples. Weiss et al. (19) show that the presence of apyrase has little effect on the observed rate of the dissociation reaction. This procedure allows the same sample to be reused a number of times with different flash intensities to vary the [ATP] released.
15 l of the extracted myosin solution in experimental buffer were mixed with 5 l of reaction mix to give a final volume of 20 l containing ϳ0.15 M myosin, 0.5 M phalloidin-stabilized actin and 0.5 mM cATP, 10 mM DTT, 10 g/ml apyrase. As shown in Fig. 2A for a sample of MHC-IIB, irradiation by a series of laser pulses of different intensities released a range of ATP concentrations. The concentration of ATP liberated in each flash was estimated from the decay of the absorbance of 405 nm, which monitors the formation and decay of the acinitro photolysis intermediate (19,25). The same sample was used for all the transients shown in Fig. 2A, and after each transient was recorded, the sample was left for 3 min for all the ATP to be hydrolyzed before the next flash. The decrease in light scattering was described by a single exponential, and the best fit to a single exponential is superimposed in each case. The observed rate constant (k obs ) of each reaction was linearly dependent upon [ATP]. Fig. 2B shows the dependence of k obs on [ATP] for each of the four MHC isoforms isolated. In each case a linear relationship was observed, and the slope of the fitted line defined the apparent second order rate constant of the ATP-induced dissociation reaction (K 1 k ϩ2 , Scheme 1). The plots show different slopes for each isoform except for IIB and IIX, which were very similar. The pooled data from a series of measurements using at least six individual fibers for each myosin isoform are given in Table I. These show that the differences between isoforms IIX and IIB are not statistically significant, whereas all other comparisons are significant.
Determination of the ADP Affinity-The competition between ADP and ATP for the myosin nucleotide site provides a method to determine the affinity of ADP for the actomyosin complex (17). At higher concentrations, ADP will reduce the affinity of myosin for actin, and at the low protein concentra- B, example of gels used for electrophoretic determination of the amount of myosin extracted from single fibers and of the remaining amount of myosin left in the fiber after myosin extraction. Lanes in which known amounts of myosin to be used as standards were loaded are indicated as s; from left to right, the amounts loaded were 1, 2, 2.5, 3, and 3.5 g. 1e, myosin extracted from fiber 1; 1r, remaining amount of myosin left in the same fiber after myosin extraction and extracted by standard buffer (20). The same applies to lanes 2e and 2r for fiber 2 and to lanes 3e and 3r for fiber 3. The gel was a 10 -20% gradient polyacrylamide gel; Coomassie staining was used.   tions used here, this results in a loss of signal amplitude (Fig.  3A). To counteract this loss of amplitude, the protein concentrations were increased to 1 M actin and 0.75 M myosin. To have sufficient myosin for these experiments, five fibers of the same isoform content were pooled for the extraction procedure. The incubation mixtures contained hexakinase and glucose to remove ATP but leave ADP present; additionally, the myokinase inhibitor Ap 5 A was added as a precaution against interconversion of ADP and ATP.
A typical experiment is shown in Fig. 3A for MHC-IIB. The first flash in the absence of ADP shows a single exponential decay of scattering as in Fig. 2A. The amount of ATP released in each flash was calculated by analysis of the transmission changes at 405 nm as before. After allowing 5 min for the elimination of the ATP, ADP was added to the sample (maximum added volume 2 l) along with a small amount of cATP to replenish that used in the previous flash. The procedure was repeated for a series 4 -6 ADP concentrations. Single exponential light-scattering transients were observed in each measurement, with both the k obs and the amplitude of the reaction decreasing as ADP concentration increased. The decrease in k obs is predicted from Equation 1 and Scheme 1. The decrease in amplitude was because of the dilution effect and the lower affinity of actin for the myosin⅐ADP complex. The ADP concentration in each case was calculated as the sum of the ADP added and the ADP that built up through the hydrolysis of the ATP released in each flash. This procedure has been validated using the well characterized rabbit muscle myosin subfragment 1 (S1). The values of k obs were corrected according to Equation 1 for the small variations in the amount of ATP released in each flash and then plotted against the ADP concentration as shown in Fig. 3B for each myosin isoform. The data was fitted to Equation 1, and the best curve was superimposed. Each MHC isoform gave a distinct value of K AD , and pooled data from three different experiments (Table I) show the differences to be significant.
Thermodynamic coupling between actin and ADP binding to myosin (17,27) predicts that the weaker the affinity of actomyosin for ADP, the weaker the affinity of myosin⅐ADP for actin. Light-scattering measurements are not very reliable for amplitude data because of factors like micro air bubbles and dust particles. However there was a clear trend for the MHC isoforms IIX and IIB to show a more pronounced loss of the amplitude of the light-scattering transients as ADP concentration was increased compared with isoforms I and IIA. This is consistent with weaker affinity of actin for isoforms IIX and IIB in the presence of ADP. DISCUSSION To date no satisfactory overexpression system exists for mammalian skeletal muscle myosin, and bulk preparations of myosin from tissue results in isolation of mixed isoforms. The majority of single muscle fibers do, however, contain single isoforms, and we have shown that we can extract sufficient myosin from single fibers for our measurements. Using our flash photolysis method, we were able to determine the rate of ATP-induced dissociation of and the ADP affinity for the actomyosin complex for all of the four skeletal muscle isoforms of adult rat. Measurements of this kind were previously not possible with these g quantities of myosin.
We obtained ϳ2 g of pure myosin isoforms from 15-mm single fibers of the rat psoas (MHC-IIB, MHC-IIX) and soleus (MHC-I and MHC-IIA). This amount is sufficient to determine the second order rate constant of ATP-induced dissociation with the flash photolysis apparatus, whereas it would be too small an amount to obtain results from traditional stopped flow methods (24). High ADP concentrations lower the affinity of myosin for actin and decrease the dissociation signal. The amount of protein from one fiber is therefore not sufficient to measure the ADP affinity to actomyosin over a wide range of ADP concentrations. For this experiment, a higher myosin concentration is needed. Five fibers for which the same isoform type has been confirmed by electrophoresis were pooled, and the myosin was extracted. The total amount of protein is still far less than would be needed using traditional methods.
Our results for both the rate of ATP-induced dissociation of actomyosin and the ADP affinity for actomyosin are significantly different for the four isoforms with the exception of the dissociation rate constants K 1 k ϩ2 between MHCs IIX and IIB ( Table I). The values were highly reproducible, showing the effectiveness of the flash photolysis method for the measurement with small amounts of actomyosin as well as the dependence of these values on the myosin heavy chain isoform.
The value of K 1 k ϩ2 increases in the isoform order MHC-I, MHC-IIA, MHC-IIX, MHC-IIB, and the affinity of ADP for actomyosin (K AD ) weakens in the same sequence. The only exception is the MHC-IIX and IIB values for K 1 k ϩ2 , which do not differ significantly. The values for these constants change in the same sequence as the increase in shortening velocity and ATPase activity of whole fibers determined previously by Bottinelli et al. (3,4). Fig. 4 shows the correlation between V 0 and K AD and between V 0 and K 1 k ϩ2 .
It has been proposed that the net rate of detachment of actomyosin cross-bridges after completion of the power stroke limits the shortening velocity in muscle fibers. The rate of release of ADP from the cross-bridge followed by the binding of ATP and the subsequent detachment of myosin from actin control the overall rate of cross-bridge detachment. Of these two events the release of ADP has been suggested to be the rate-limiting step (10). Our data show that both the rate of ATP binding (K 1 k ϩ2 ) and the affinity of ADP (K AD ), which is related to the rate of ADP release (k ϪAD ), show good correlation with shortening velocity. Although suggestive of an important role of K 1 k ϩ2 and K AD in determining shortening velocity, such a correlation does not establish whether either one of the steps actually determines shortening velocity alone or the relative contribution of each of them. To evaluate more carefully the contribution of these events to shortening velocity, we must estimate K 1 k ϩ2 and K AD values under the more physiological conditions used to measure shortening velocities and quantitatively compare them. This will also allow the comparison of k ϪAD , which, we will suggest, can limit shortening velocity, with the value of k min (rate of the event limiting shortening velocity) estimated from the maximum shortening velocity of single muscle fibers (10).
In our experimental setup the binding of ADP to actomyosin can be described as a fast equilibrium with rate constants ([ADP]⅐k ϩAD ϩ k ϪAD ) more than 10 times faster than the rate of the following ATP binding and dissociation ([ATP]⅐K 1 k ϩ2 ). Under those conditions the net rate of detachment is determined by Equation 1, which remains valid for all of our measurements where the [ATP] is Ͻ100 M. In the cell the [ATP] is much higher, and this fast equilibrium assumption for ADP binding may no longer be valid. Estimates of the [ATP] in intact muscle fiber are in the range 4 -8 mM (28), and 5 mM was used in the V 0 measurements.
Our values were measured in solution using whole myosin and were therefore made at a KCl concentration of 0.5 M to keep the myosin soluble. Both K 1 k ϩ2 and K AD values need to be corrected for the ionic strength in the fiber, which was 0.2 M in the V 0 measurements (4). In principle it would be possible to repeat the measurements with HMM or S1 and to work at physiological ionic strength. However, because of the very small amounts of myosin we have available, digestion of the protein would lead to unacceptable losses of protein. The corrections necessary can be estimated from a Debye-Hü ckel plot for K AD and K 1 k ϩ2 obtained using bulk preparations of skeletal myosin S1. Such measurements have been made for S1 from the back and leg muscle of the rabbit and for S1 isolated from rat soleus and edl muscle. 2 The Debye-Hü ckel plot (not shown) for K AD shows a 3-7-fold decrease from 0.5 to 0.1 M KCl that corresponds to an ionic strength of ϳ0.2 M. For the same change of ionic strength K 1 k ϩ2 is increased about 3-5-fold (29). The effect of these corrections is shown in Table II Table  I multiplied by 2 (in segment length, L, per second). The rates of dissociation of actomyosin as physiological salt conditions were calculated from the values of K 1 k ϩ2 in Table I   The rate of ADP binding to (k ϩAD ) and dissociation from (k ϪAD ) actomyosin are too fast to be measured using the method described here. At the limit we know the sum ([ADP]⅐k ϩAD ϩ k ϪAD ) is much greater than [ATP]⅐K 1 k ϩ2 , which has measured values of up to 10 s Ϫ1 (see Fig. 2). Siemankowski and White (17) calculated the binding rate of ADP to actomyosin, k ϩAD (from measured values of K AD and k ϪAD ), to be 10 7 M Ϫ1 s Ϫ1 . This is close to the value expected for a diffusionlimited process. If we assume that ADP binding is a diffusionlimited process and is therefore similar for all myosins, then we can estimate the dissociation rate constant from k ϪAD ϭ K AD ⅐k ϩAD . Thus, taking the corrected values of K AD , we can calculate the values of k ϪAD at physiological ionic strength to be about 600 s Ϫ1 , 1150, 1790, and 2220 s Ϫ1 (with the range of possible values shown in Table II) for the isoforms MHC-I, MHC-IIA, MHC-IIX, and MHC-IIB, respectively.
Siemankowski et al. (10) propose that the minimum value of the rate constant (k min ) of the event limiting maximum shortening velocity (V 0 ) can be estimated from the half-sarcomere length (S L ) and the maximum allowed axial cross-bridge translation (d, Equation 2). The values of V 0 from Table I were measured at 12°C. The Q 10 for V 0 in rat muscles is ϳ2 (30), which predicts that values will be doubled at 20°C, which is the temperature used in the kinetic measurements presented here (corrected V 0 values given in Table II). The value of the half-sarcomere length (1.35 m) is that of the fibers used in the V 0 measurements by Bottinelli et al. (4), and the step size is assumed to be 5-10 nm. With these values, k min can be calculated, and the result is shown in Table II. Even within the limits of the measurements and corrections made here, the agreement between the values of k min and k ϪAD is remarkably close for all four myosin isoforms, less than a factor of 2 in each case. The results are therefore compatible with the rate of ADP release limiting the shortening velocity for the four muscle fibers as predicted by Siemankowski et al. (10).
Under physiological conditions the concentration of ATP is of the order of 5 mM; thus, the rates of ATP binding and actin dissociation, [ATP]⅐K 1 k ϩ2 , are approximated as shown in Table  II. Taking the mid-range values of [ATP]⅐K 1 k ϩ2 , these are ϳ4fold faster than k min for all MHC-II isoforms and 8-fold faster for MHC-I. Thus, it is clear that [ATP]⅐K 1 k ϩ2 is unlikely to contribute to k min for the MHC-I isoform, but the situation is not clear cut for the faster isoforms. At the lower limits of the estimated range, the ATP binding step could be a significant contributor to k min and is of the same order as k ϪAD . It is of interest to note that differences in K 1 k ϩ2 [ATP] and k ϪAD are smallest for the fastest MHC-IIB (2.5-fold) and largest for the slow MHC-I (5.5-fold). Thus, any significant drop in the concentration of ATP (or an increase in the competing free [ADP]) could result in a slowing of A⅐M dissociation and lowering of V 0 . Interestingly the maximum shortening velocity (V 0 ) of fast fibers has been shown to be more affected by a decrease in [ATP] than the V 0 of slow fibers (31). It is possible that this is a contributor to fatigue-induced loss of muscle performance, which is more marked in fast muscles.
This result demonstrates that for four closely related muscle myosins, the shortening velocity is controlled by the rate at which ADP can escape from the cross-bridge after completion of the power stroke. A contribution from ATP dissociation of the cross-bridge remains possible for the fastest isoforms. It is of interest to note the ATP-induced dissociation varies by less than 2-fold between the four myosin types compared with a 3.5-fold variation in both k min and k ϪAD . The faster ATP-induced dissociation rate for the faster myosins has previously been reported for different muscle types of chicken muscle (9). It appears that this step must remain faster than the preceding ADP release step, but its precise value does not correlate as closely with the shortening velocity. It may not be surprising that both ATP binding and K AD show a similar (but not identical) dependence on myosin isoform since both nucleotides are binding to the same pocket. Thus, changes in sequence which result in changes of the biochemical environment of the nucleotide binding pocket that lead to a faster ADP release may also produce faster binding of ATP.
Several comparisons between myosin isoforms have highlighted Loop1 (on the myosin surface at the entrance to the nucleotide pocket) as responsible for modulating ADP affinity to actomyosin and velocity of either the muscle fiber shortening or in in vitro motility assays (11). These include scallop-striated and catch muscle myosin (32) and the phasic and tonic isoforms of smooth muscle myosin (12,13) in addition to artificial myosin constructs (33). It is therefore of interest to consider the changes in sequence that produce these changes in the properties of the rat striated muscle myosin isoforms. Unfortunately the sequences of the MHC-II rat isoforms are not currently in the data base; only the MHC-I is known. However, given that the MHC-I sequences are very heavily conserved between the rat and human, a similar conservation may be expected in the MHC-II sequences. The human sequences are all known and have been compared by Weiss et al. (34). These show ϳ90% identity between the three MHC-II sequences and 80% identity comparing the MHC-I and II. Surprisingly, although there are changes in sequences in Loop 1 between MHC-I and -II, the sequence of Loop I is well conserved among the three MHC-II myosins, and it may be necessary to look for more subtle changes in structure to account for the 2-fold changes in V 0 and K AD reported here.
In conclusion we have confirmed the hypothesis proposed by Siemankowski et al. (10) for this closely related set of rat MHC isoforms in that ADP release can provide the limiting molecular event that limits shortening velocity. We have estimated that under normal conditions the rate of the ATP-induced dissociation of A⅐M remains up to 2-6-fold faster than the preceding step of ADP release from A⅐M⅐D. Further information on the sequence differences between the rat MHC isoforms will be required to understand how the ADP release rate is modulated by the myosin structure. Alternatively a similar study on the equivalent human isoforms for which sequence information is known will be of interest, and we are attempting such a study.