©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
In Vitro Motility Analysis of Smooth Muscle Caldesmon Control of Actin-Tropomyosin Filament Movement (*)

(Received for publication, March 20, 1995; and in revised form, May 15, 1995)

Iain D. C. Fraser Steven B. Marston (§)

From the Department of Cardiac Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

We have used the in vitro motility assay to investigate the effect of caldesmon on the movement of actin-tropomyosin filaments over thiophosphorylated smooth muscle myosin and skeletal muscle heavy meromyosin. Using either motor, incorporation of up to 8 nM caldesmon inhibited filament movement by decreasing the proportion of filaments motile from >85% to <30%. There was a minimal effect on filament attachment and a modest decrease in motile filament velocity in this concentration range. The reduction in the proportion of filaments motile could be completely reversed by incorporation of an excess of calmodulin at pCa 4.5. The expressed C-terminal fragment, 606C, which retains caldesmon's inhibitory capacity but does not bind to myosin, decreased the proportion of filaments motile but had no effect on velocity. We conclude that the velocity reduction by whole caldesmon is due to actin-myosin cross-linking. A significant decrease in filament attachment was observed when caldesmon was added to an excess over actin (>10 nM). In the absence of tropomyosin, addition of an excess of caldesmon caused a similar decrease in the filament density, but there was no effect on the proportion of filaments that were motile. Our results demonstrate that caldesmon can switch actin-tropomyosin from motile to non-motile states without controlling velocity of movement or weak binding affinity and show the inhibitory action of caldesmon in the motility assay to be functionally indistinguishable from that reported for troponin.


INTRODUCTION

Smooth muscle actomyosin is regulated by Ca through phosphorylation of myosin by the Ca-calmodulin-dependent myosin light chain kinase(1) . However, the thin filaments are also Ca regulated(2) , and there is a growing body of evidence that a parallel regulation of the thin filament of smooth muscle, mediated by the protein caldesmon(3, 4, 5, 6) , plays some role in controlling contractility in intact smooth muscle.

Caldesmon has been shown to be a potent inhibitor of the actin-tropomyosin-activated myosin ATPase in vitro(7, 8, 9, 10) . One caldesmon molecule can inhibit 14 actin monomers in the presence of smooth muscle tropomyosin, and this inhibition is reversed by Ca in the presence of a Ca binding protein such as calmodulin(7, 11) .

The mechanism by which caldesmon controls the smooth muscle thin filament has been closely studied. Caldesmon can inhibit the actin-activated ATPase by competitive blocking of the myosinbulletADPbulletP(i) ``weak'' interaction with actin, and this has been proposed as a means of regulation(12, 13) . This, however, would require a stoichiometry of 1 caldesmon:1 actin, which would be incompatible with the ratio of caldesmon to actin in the contractile domain of the smooth muscle cell and in native thin filaments(14, 15, 16) . In the presence of smooth muscle tropomyosin, caldesmon can inhibit the ATPase activity at much lower caldesmon:actin ratios(7, 14, 15) . In this case, caldesmon is proposed to act as an allosteric effector, switching the thin filament between ``on'' and ``off'' states in much the same way as troponin does in striated muscles(15, 17) .

The in vitro motility assay, developed by Kron and Spudich (18) , provides an ideal method to investigate the regulation of individual thin filaments, and several workers have studied the effect of caldesmon. Okagaki et al.(19) incorporated caldesmon by pre-mixing with actin and tropomyosin and found that the average velocity of filaments taken over a 20-s period was progressively reduced by increasing caldesmon up to a 5-fold excess over actin. Shirinsky et al.(20) , with a similar caldesmon:actin ratio, also noted a decrease in filament velocity. They infused caldesmon and tropomyosin into the assay cell after the labeled actin. They also noted that the proportion of stationary filaments was increased and that ``apparent'' filament velocity could be decreased by erratic ``stop-start'' movement. Haeberle et al.(21) , working at a higher ionic strength, infused caldesmon up to a 100-fold excess over actin and noted no effect on the velocity of continuously motile actin filaments. Their selective analysis, however, excluded filaments exhibiting ``stop-start'' motion, which is likely to have contributed to the velocity reduction observed in the other studies.

Recently, we analyzed troponin control of actin-tropomyosin filament motility and found that troponin, in the absence of Ca, reduced the proportion of filaments that were motile without changing the velocity or the number of filaments attached to the myosin(22) .

Since the disagreement in the results with caldesmon appears to be due to the various methods of selective analysis, we have investigated the control of thin filament motility by caldesmon using the criteria established in our study of troponin. 1) Data are only collected from experiments where at least 80% of filaments are motile in control assays. 2) Filaments are randomly selected for tracking prior to analysis to avoid any bias in the collection of data. 3) A complete analysis of filament movement is carried out on a frame-to-frame basis. This involves determination of the proportion of non-motile filaments, the velocity of motile filaments, and the average number of filaments attached to the immobilized motor.

On this basis, we have carried out a detailed analysis of our assays to determine a mechanism by which caldesmon controls actin-tropomyosin filament movement over thiophosphorylated smooth muscle myosin and skeletal muscle HMM. (^1)In both cases, low concentrations of caldesmon primarily decreased the proportion of filaments that were motile without significantly affecting the filament density. A modest decrease in the filament velocity mediated by whole caldesmon was not observed when using an expressed fragment (606C) comprising the C-terminal 150 amino acids of caldesmon, which does not bind myosin. The reduction in the proportion of filaments motile by caldesmon was fully reversed by an excess of calmodulin at pCa 4.5.


MATERIALS AND METHODS

Preparation of Proteins

Rabbit skeletal muscle actin and chicken gizzard smooth muscle tropomyosin, caldesmon, and crude myosin were prepared by published methods(7, 23) . Rabbit skeletal muscle HMM was prepared from myosin, and skeletal muscle f-actin was labeled with rhodamine phalloidin () as described by Kron et al.(24) . The recombinant caldesmon fragment 606C was prepared by expression in Escherichia coli and purified as previously described(25) . Calmodulin from bovine brain was prepared according to (26) .

Crude smooth muscle myosin (3 mg/ml), containing endogenous myosin light chain kinase, was thiophosphorylated by incubation with 0.3 µM calmodulin and 1 mM ATPS in 60 mM KCl, 25 mM imidazole-HCl (pH 7.4), 10 mM MgCl(2), 0.2 mM CaCl(2), 1 mM dithiothreitol at 25 °C for 20 min.

In Vitro Motility Assay

Actinbulletbullettropomyosin and actinbulletbullettropomyosinbulletcaldesmon complexes were mixed at 10 assay concentration for 30-60 min and diluted immediately prior to infusion into the assay cell as previously described(22) . Final assay concentrations for f-actinbullet and smooth muscle tropomyosin were 10 and 40 nM, respectively.

Thiophosphorylated smooth muscle myosin and skeletal muscle HMM were pre-treated to minimize the presence of ``rigor heads'' as previously described(22) .

All experiments were carried out using coverslips coated with silicone by soaking in 0.2% dichloromethylsilane in chloroform. A flow cell was prepared from a freshly siliconized coverslip and a microscope slide as described by Kron et al.(24) . Assay components and buffers were infused into the flow cell at 30-60-s intervals. Two 50-µl aliquots of skeletal muscle HMM at 100 µg/ml were infused in buffer A (50 mM KCl, 25 mM imidazole-HCl, 4 mM MgCl(2), 1 mM EDTA, 5 mM dithiothreitol, pH 7.4) to provide a coating of immobilized HMM on the coverslip. This was followed by 2 50 µl of buffer B (A + 0.5 mg/ml bovine serum albumin) then 2 50 µl of 10 nM actinbullet ± associated tropomyosin-caldesmon in buffer A. 50 µl of buffer C (B + 0.1 mg/ml glucose oxidase, 0.02 mg/ml catalase, 3 mg/ml glucose, 0.5% methylcellulose ± caldesmon at assay concentration) and 50 µl of buffer D (C + 1 mM ATP) were then infused.

Smooth muscle myosin was infused into the assay cell in monomeric form in 300 mM KCl, and this KCl concentration was maintained for the wash with bovine serum albumin containing buffer (buffer B). Otherwise, the assay procedure was as described above for skeletal muscle HMM.

The rhodamine fluorescence of actinbullet filaments was observed using a Zeiss epifluorescence microscope (63/1.25 objective) with a DAGE-SIT-68 camera and recorded on video tape. Sequences of video images were digitized through a frame grabber, installed on a Macintosh IIci computer, at 0.65-s (skeletal HMM) or 3.65-s (smooth myosin) intervals.

Analysis of Filament Movement

The movement of actinbullet filaments was analyzed, and the density of filaments attached to the immobilized substrate was determined as previously described(22) . Data were only collected from assay cells in which at least 80% of actinbullet or actinbulletbullettropomyosin filaments were motile.


RESULTS

The potentiating effect of smooth muscle tropomyosin on the actin-activated myosin ATPase is well documented(27, 28) . We found that introducing tropomyosin into the assay at 0.085 ionic strength, 28 °C, increased the velocity of actin filaments over skeletal muscle HMM as shown by Umemoto and Sellers(29) . We noted, however, that the method of introduction was important. If tropomyosin was simply infused into the assay cell after the actin filaments, the level of velocity potentiation at increasing tropomyosin concentrations was inconsistent. If, however, we pre-mixed the actin and tropomyosin at 10 assay concentrations and diluted the proteins prior to use, the filament velocity increased in a regular manner with increasing tropomyosin concentration (Fig. 1). We chose 40 nM tropomyosin as a control concentration for subsequent assays, and binding of tropomyosin to actin at this concentration is directly demonstrated by cosedimentation and SDS-gel electrophoresis (Fig. 1, inset). Using thiophosphorylated smooth myosin, incorporation of 40 nM tropomyosin at 28 °C increased the velocity of actin filaments from 0.54 to 0.71 µm/s.


Figure 1: The effect of smooth muscle tropomyosin on actinbullet filament velocity. F-actinbulletbullettropomyosin complexes were formed at a range of tropomyosin concentrations, and the velocity of filaments was analyzed. Datapoints represent mean velocities with standard errors from 20 filaments. Filament velocity increases sharply with incorporation of tropomyosin to 40 nM. Inset, binding of tropomyosin to actinbullet at motility assay conditions. Samples of undiluted (100 nM actin + 400 nM tropomyosin) and 10-fold diluted actinbulletbullettropomyosin complexes were centrifuged for 30 min at 150,000 g to sediment actinbullet filaments. SDS-gel electrophoresis of the pellets is shown as a digitized image. Tropomyosin was bound to the actinbullet in both the undiluted (A) and the 10-fold diluted (B) samples, respectively.



Effect of Caldesmon on Filament Movement over Skeletal Muscle HMM

Caldesmon was introduced into the motility assay as described by pre-mixing with actin and tropomyosin.

On observation of assays with increasing caldesmon concentration, it was clear that the principal effect of caldesmon was to increase the proportion of stationary actin-tropomyosin filaments.

To analyze this quantitatively, we collected sequences of 11 video images at 0.65-s intervals from each assay. To avoid bias in selection of filaments for analysis, we chose 10 filaments at random from a printout of the first image prior to tracking.

Fig. 2shows the tracks of filaments at increasing caldesmon concentrations. It is clear from the tracks that have less than 11 ``points'' that filaments have an increased tendency to exhibit ``stop-start'' motion in the presence of caldesmon. This would result in an apparent decrease in velocity for these filaments if we calculated velocity purely from displacement over an 11-frame sequence. To avoid this false interpretation, we measured the 10 individual frame-to-frame velocities for each filament. From these data, it was clear that there were two populations of filaments: one with zero velocity, which we classified as non-motile, and another with velocities of 3.5-5 µm/s, which we classified as motile. Instances where filaments stopped or started between frames of the tracking sequence typically comprised 5-10% of total data points in the presence of caldesmon. This could give an intermediate velocity for that step; however, when we left out these data points, there was no significant difference in the average motile filament velocity.


Figure 2: The effect of caldesmon on actinbulletbullettropomyosin movement over rabbit skeletal muscle HMM. The tracks of 10 filaments from six separate assays with increasing caldesmon concentration are shown. The points in each track represent the filament position at 0.65-s intervals through 11 frames. Tracks with fewer than 11 points are from filaments that stopped or started during the tracking sequence; examples are indicated by arrows. Immobile filaments appear as singledots and are highlighted with graycircles.



Fig. 3A demonstrates that the proportion of filaments that were motile decreases sharply with increasing caldesmon concentration. We also determined the velocity of motile filaments and the average number of filaments attached to the immobilized HMM. Fig. 3B demonstrates that there is a gradual decrease in velocity from 4.9 to 3.7 µm/s, but filament density shows little change over the range of caldesmon concentrations required to reduce the proportion of filaments motile to 20% (Fig. 3A). Filament density is seen to decline at the higher caldesmon concentrations.


Figure 3: Quantitative analysis of the effect of caldesmon and calmodulin on actinbulletbullettropomyosin filament motility. Frame-to-frame velocities were calculated for each filament, 10 values per 11-frame sequence, and the proportion of filaments moving and mean velocity of motile filaments were calculated. Datapoints represent means ± S.E. from 40-60 filaments taken from separate experiments. Filament density was determined as described under ``Materials and Methods.'' A, proportion of filaments motile (bullet) and filament density () at increasing caldesmon concentrations. B, velocity of motile filaments (diamond, filled) at increasing caldesmon concentrations. C, proportion of filaments motile (bullet) and filament density () at increasing calmodulin concentrations (caldesmon fixed at 6 nM, pCa 4.5). Proportion of filaments motile (up triangle, filled) with 120 nM calmodulin at pCa 9.0. D, velocity of motile filaments (diamond, filled) at increasing calmodulin concentrations (caldesmon fixed at 6 nM, pCa 4.5).



Ca-calmodulin was introduced into the assay to determine its effect on the actin-tropomyosin-caldesmon filaments. Using 10 nM actin, 40 nM tropomyosin, and 6 nM caldesmon, increasing calmodulin concentrations from 20 to 120 nM were investigated. Fig. 3, C and D, shows the only effect of Ca-calmodulin was to increase the proportion of filaments motile from 25.5 to 83.5% with no effect on the number of filaments attached to the HMM or the velocity of motile filaments. Addition of 120 nM calmodulin at low Ca (pCa 9.0) had no effect on the motile proportion, velocity, or density of filaments (Fig. 3C).

It has been shown previously that the inhibitory properties of caldesmon are located in the C-terminal region, represented by the 20-kDa fragment (30, 31) or the expressed recombinant, 606C, comprising the C-terminal 150 amino acids(606-756) of chicken gizzard caldesmon (25) . In the motility assay, incorporation of up to 200 nM 606C reduced the proportion of filaments motile from 84.5 to 22.3% with little or no effect on filament density (Fig. 4A). In contrast to the slight velocity reduction seen with whole caldesmon, 606C had no appreciable effect on the velocity of motile filaments (Fig. 4B).


Figure 4: The effect of 606C on actinbulletbullettropomyosin filament motility. A, proportion of filaments motile (bullet) and filament density (). B, velocity of motile filaments (⧫). Calculations were carried out as described for Fig. 3. Datapoints represent means ± S.E. from 40 filaments taken from separate experiments.



Further experiments were carried out using caldesmon pre-mixed with actin in the absence of tropomyosin. The same concentration range of 2-15 nM caldesmon had minimal effect on the proportion of filaments motile or the velocity of motile filaments (Fig. 5, A and B). The filament density, however, was constant with addition of up to 6 nM caldesmon (Fig. 5A) before declining at higher caldesmon concentrations in much the same way as observed in the corresponding experiments where tropomyosin was present.


Figure 5: The effect of caldesmon on actinbullet filament motility in the absence of tropomyosin. A, proportion of filaments motile (bullet) and filament density (). B, velocity of motile filaments (⧫). Calculations were carried out as described for Fig. 3. Datapoints represent means ± S.E. from 40 filaments taken from separate experiments.



Effect of Caldesmon on Filament Movement over Thiophosphorylated Smooth Muscle Myosin

Motility assays were also carried out using smooth muscle myosin as the immobilized motor. The effect of caldesmon on filament motility is shown in Fig. 6, A and B. As observed using skeletal muscle HMM, the primary action of caldesmon was to reduce the proportion of filaments motile from 85.8 to 13.5% with minimal effect on the filament density at the lower caldesmon concentrations (Fig. 6A). Similarly, there was a gradual reduction in the velocity of motile filaments from 0.67 to 0.41 µm/s (Fig. 6B).


Figure 6: The effect of caldesmon and 606C on actinbulletbullettropomyosin filament motility using thiophosphorylated smooth muscle myosin. A and B, proportion of filaments motile (bullet), filament density (), and velocity of motile filaments (⧫) at increasing caldesmon concentrations. C and D, proportion of filaments motile (bullet), filament density (), and velocity of motile filaments (⧫) at increasing 606C concentrations. Calculations were carried out as described for Fig. 3. Datapoints represent means ± S.E. from 40 filaments taken from separate experiments.



Incorporating the C-terminal fragment 606C also gave a significant reduction in the proportion of filaments motile but no effect on motile filament velocity (Fig. 6, C and D). Filament density was again constant with addition of up to 200 nM 606C before declining at the highest 606C concentration of 300 nM (Fig. 6C).

When tropomyosin was absent, the proportion of filaments motile and the velocity of motile filaments were not significantly reduced by caldesmon. The filament density was constant up to 6 nM caldesmon before declining at the highest caldesmon concentrations (data not shown).


DISCUSSION

We have used the in vitro motility assay to determine how caldesmon may inhibit contractility in the smooth muscle cell. Other studies have addressed the question, but the methods of introduction of caldesmon into the assay have varied, as have the assay conditions and the concentrations of caldesmon used(19, 20, 21, 32) .

In a recent study with troponin(22) , we found the most consistent method, when incorporating thin filament proteins into the assay, was to pre-mix at 10 assay concentrations and then dilute immediately prior to use. This presumably improves the binding efficiency at the very low concentrations employed in the assay. We also found it was important to analyze filaments chosen at random and to determine velocity over a short ``frame-to-frame'' time scale, since filaments could stop and start during the assay.

We have used the same techniques to study the effect of low concentrations of caldesmon on the motility of actin-tropomyosin filaments. Using either skeletal muscle HMM or thiophosphorylated smooth muscle myosin as the immobilized motor, incorporation of up to 6 nM caldesmon sharply reduced the proportion of actin-tropomyosin filaments motile. As there was no significant effect on the density of filaments attached and only a modest effect on filament velocity in this concentration range, it would appear that caldesmon reduces the proportion of actin-tropomyosin filaments that are motile in much the same way as observed with troponin(22) . Examination of the data of Shirinsky et al.(20) , Haeberle et al.(21) , and Horiuchi and Chacko (32) indicates that similar effects have been observed before, but their significance has not been recognized.

It is widely accepted that the inhibitory effect of caldesmon is reversed by Ca-calmodulin in vitro(7, 11) . It is therefore significant to note the effect of Ca-calmodulin on the motility of actin-tropomyosin-caldesmon filaments in the motility assay. Increasing calmodulin concentrations at pCa 4.5 had no effect on the velocity of motile filaments or the filament density, but a 20-fold excess of calmodulin over caldesmon completely reversed the reduction in the proportion of filaments motile back to the level observed with actin-tropomyosin alone. This indicates that the reduction in % motility was the principal mechanism of the inhibition mediated by caldesmon. Caldesmon was effective at low concentrations, indicating an apparent affinity of >10^8M in the presence of tropomyosin. This corresponds to the high affinity-low stoichiometry, tropomyosin-dependent component of caldesmon binding to actin-tropomyosin, first demonstrated by Smith et al.(7) (K 10^8M) and subsequently by others(25, 33, 34) .

The experiments carried out with the C-terminal fragment, 606C, provide additional information. This fragment contains residues 606-756 of chicken gizzard caldesmon and is a tropomyosin-dependent, Ca-calmodulin-regulated inhibitor of the actomyosin ATPase(25) . Unlike whole caldesmon, 606C does not bind to myosin. In our assays with skeletal HMM and smooth myosin, 606C reduced the proportion of filaments motile without displacing them from the motor until the highest concentrations, in much the same way as whole caldesmon. This demonstrates that the increase in non-motility observed with whole caldesmon is not due to filaments being displaced from myosin and then ``tethered'' through caldesmon's joint interaction with actin and myosin.

The decrease in the velocity of motile filaments seen using whole caldesmon is not observed using 606C. This shows that the velocity reduction is due to the N-terminal myosin binding site of caldesmon imposing a mechanical load on motile filaments, as proposed by Shirinsky et al.(20) and more recently by Horiuchi and Chacko(32) . This is further supported by our assays that show a more significant velocity reduction when using smooth muscle myosin as the immobilized motor, due to its stronger interaction with caldesmon(35, 36) .

The extent to which velocity is reduced by caldesmon would consequently be dependent on ionic strength, as the interaction of caldesmon's N terminus with myosin has been shown to be salt dependent(36, 37) . It is not surprising therefore that the velocity reduction observed by Shirinsky et al.(20) with 25 mM KCl is more significant than we observe using 50 mM KCl, where the caldesmon-myosin interaction is weaker, while at 80 mM KCl, Haeberle et al.(21) observe no significant effect on velocity from added caldesmon.

Inhibition of the actomyosin ATPase by low concentrations of caldesmon has been shown to be highly dependent on smooth muscle tropomyosin(7, 15) . We observed a similar tropomyosin dependence in our assays in caldesmon's ability to ``switch off'' motile filaments, where, in the absence of tropomyosin, up to 15 nM caldesmon had little effect on the proportion of filaments motile. The filament density, however, began to gradually decrease at the higher caldesmon concentrations in the presence or absence of tropomyosin. This demonstrates that direct displacement of actin filaments from myosin is a tropomyosin-independent phenomenon, most evident when caldesmon is present in a molar excess over actin. This effect was also reported by Haeberle et al.(21) , when complete filament displacement was observed on addition of a C-terminal fragment of caldesmon to a 240-fold excess over actin.

The effect of caldesmon observed in the motility assay can be used to gain insight into how caldesmon may operate to regulate the smooth muscle thin filament in vivo. The ``power stroke'' in muscle contraction is dependent on the transition from a weak binding complex between actin and myosin to a strong binding complex with subsequent release of ADP and P(i)(38, 39, 40) . As low concentrations of caldesmon can switch off actin-tropomyosin filaments without dissociating them from myosin, it would appear that inhibited filaments remain bound to the immobilized HMMbulletADPbulletP(i) via weak interactions. Displacement of the thin filaments from HMMbulletADPbulletP(i) is independent of tropomyosin and is observed at higher caldesmon concentrations when there is a sufficient number of caldesmon molecules to bind to every actin monomer(15) . This is unlikely to occur in vivo, as there is only one caldesmon for every 14 actin monomers in the smooth muscle thin filament(11, 15, 16) .

Caldesmon therefore switches off actin filaments by acting on tropomyosin to block the strong binding myosin complexes, as recently proposed from solution experiments(17) , suggesting that caldesmon controls actin-tropomyosin filaments as a cooperative unit in smooth muscle. Recent studies with striated muscle thin filaments in the motility assay(22) , demonstrating an analogous control mechanism with troponin, show that the two proteins have important similarities in function and in mechanism of action.


FOOTNOTES

*
This work was supported by the British Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-171-352-8121 (ext. 3307); Fax: 44-171-823-3392; S.Marston{at}ucl.ac.uk or I.Fraser{at}ucl.ac.uk.

(^1)
The abbreviations used are: HMM, heavy meromyosin; , rhodamine-phalloidin; ATPS, adenosine 5`-O-(thiotriphosphate).


REFERENCES

  1. Murphy, R. A. (1994) FASEB J. 8,311-318 [Abstract/Free Full Text]
  2. Marston, S. B., and Smith, C. W. (1985) J. Musc. Res. Cell Motil. 6,669-708 [Medline] [Order article via Infotrieve]
  3. Barany, M., and Barany, K. (1993) Arch. Biochem. Biophys. 305,202-204 [CrossRef][Medline] [Order article via Infotrieve]
  4. Gerthoffer, W. T. (1987) J. Pharmacol. Exp. Ther. 240,8-15 [Abstract]
  5. Khalil, R. A., Wang, C-L. A., and Morgan, K. G. (1995) Biophys. J. 68,75 (abstr.)
  6. Pfitzer, G., Zeugner, C., Trotschka, M., and Chalovich, J. M. (1993) Proc. Natl. Acad. Sci. U. S. A. 90,5904-5908 [Abstract]
  7. Smith, C. W., Pritchard, K., and Marston, S. B. (1987) J. Biol. Chem. 262,116-122 [Abstract/Free Full Text]
  8. Dabrowska, R., Goch, A., Galazkiewicz, B., and Osinska, H. (1985) Biochim. Biophys. Acta 842,70-75 [Medline] [Order article via Infotrieve]
  9. Sobue, K., Morimoto, K., Inui, M., Kanda, K., and Kakiuchi, S. (1982) Biomed. Res. 3,188-196
  10. Ngai, P. K., and Walsh, M. P. (1984) J. Biol. Chem. 259,13656-13659 [Abstract/Free Full Text]
  11. Marston, S. B., and Redwood, C. S. (1991) Biochem. J. 279,1-16 [Medline] [Order article via Infotrieve]
  12. Hemric, M. E., and Chalovich, J. M. (1988) J. Biol. Chem. 263,1878-1885 [Abstract/Free Full Text]
  13. Chalovich, J. M., Hemric, M. E., and Velaz, L. (1990) Ann. N. Y. Acad. Sci. 599,85-99 [Medline] [Order article via Infotrieve]
  14. Marston, S. B., and Redwood, C. S. (1992) J. Biol. Chem. 267,16796-16800 [Abstract/Free Full Text]
  15. Marston, S. B., and Redwood, C. S. (1993) J. Biol. Chem. 268,12317-12320 [Abstract/Free Full Text]
  16. Marston, S. B. (1990) Biochem. J. 272,305-310 [Medline] [Order article via Infotrieve]
  17. Marston, S. B., Fraser, I. D. C., and Huber, P. A. J. (1994) J. Biol. Chem. 269,32104-32109 [Abstract/Free Full Text]
  18. Kron, S. J., and Spudich, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,6272-6276 [Abstract]
  19. Okagaki, T., Higahi-Fujime, S., Ishikawa, R., Takano-Ohmuro, H., and Kohama, K. (1991) J. Biochem. (Tokyo) 109,858-866 [Abstract]
  20. Shirinsky, V., Biryukov, K. G., Hettasch, J. M., and Sellers, J. R. (1992) J. Biol. Chem. 267,15886-15892 [Abstract/Free Full Text]
  21. Haeberle, J. R., Trybus, K. M., Hemric, M. E., and Warshaw, D. M. (1992) J. Biol. Chem. 267,23001-23006 [Abstract/Free Full Text]
  22. Fraser, I. D. C., and Marston, S. B. (1995) J. Biol. Chem. 270,7836-7841 [Abstract/Free Full Text]
  23. Sellers, J. R., Pato, M. D., and Adelstein, R. S. (1981) J. Biol. Chem. 256,13137-13142 [Abstract/Free Full Text]
  24. Kron, S. J., Toyoshima, Y. Y., Uyeda, T. Q. P., and Spudich, J. A. (1991) Methods Enzymol. 196,399-416 [Medline] [Order article via Infotrieve]
  25. Redwood, C. S., and Marston, S. B. (1993) J. Biol. Chem. 268,10969-10976 [Abstract/Free Full Text]
  26. Gopalakrishna, R., and Anderson, W. B. (1982) Biochem. Biophys. Res. Commun. 104,830-836 [Medline] [Order article via Infotrieve]
  27. Williams, D. L., Greene, L. E., and Eisenberg, E. (1984) Biochemistry 23,4150-4155 [Medline] [Order article via Infotrieve]
  28. Lehrer, S. S., and Morris, E. P. (1984) J. Biol. Chem. 259,2070-2072 [Abstract/Free Full Text]
  29. Umemoto, S., and Sellers, J. R. (1990) J. Biol. Chem. 265,14864-14869 [Abstract/Free Full Text]
  30. Szpacenko, A., and Dabrowska, R. (1986) FEBS Lett. 202,182-186 [CrossRef][Medline] [Order article via Infotrieve]
  31. Leszyk, J., Mornet, D., Audemard, E., and Collins, J. H. (1989) Biochem. Biophys. Res. Commun. 160,1371-1378 [Medline] [Order article via Infotrieve]
  32. Horiuchi, K. Y., and Chacko, S. (1995) J. Muscle. Res. Cell Motil. 16,11-19 [Medline] [Order article via Infotrieve]
  33. Moody, C. J., Marston, S. B., and Smith, C. W. (1985) FEBS Lett. 191,107-112 [CrossRef][Medline] [Order article via Infotrieve]
  34. Yamakita, Y., Yamashiro, S., and Matsumura, F. (1992) J. Biol. Chem. 267,12022-12029 [Abstract/Free Full Text]
  35. Marston, S. B., Pinter, K., and Bennett, P. M. (1992) J. Musc. Res. Cell Motil. 13,206-218 [Medline] [Order article via Infotrieve]
  36. Hemric, M. E., and Chalovich, J. M. (1990) J. Biol. Chem. 265,19672-19678 [Abstract/Free Full Text]
  37. Bogatcheva, N. V., Vorotnikov, A. V., Birukov, K. G., Shirinsky, V. P., and Gusev, N. B. (1993) Biochem. J. 290,437-442 [Medline] [Order article via Infotrieve]
  38. Rayment, I., and Holden, H. (1994) Trends. Biochem. Sci. 19,129-134 [Medline] [Order article via Infotrieve]
  39. Lehrer, S. S. (1994) J. Musc. Res. Cell Motil. 15,232-236 [Medline] [Order article via Infotrieve]
  40. Chalovich, J. M., Yu, L. C., and Brenner, B. (1991) J. Musc. Res. Cell Motil. 12,503-506 [Medline] [Order article via Infotrieve]

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