Biochemical and Biophysical Research Communications 272, 586 –590 (2000)
doi:10.1006/bbrc.2000.2819, available online at http://www.idealibrary.com on

Direct Observation of Processive Movement
by Individual Myosin V Molecules
Takeshi Sakamoto,* Ichiro Amitani,* Etsuo Yokota,† and Toshio Ando* ,1*Department of Physics, Faculty of Science, Kanazawa University, Kakuma-machi, Kanazawa, Ishikawa-ken 920-1192,
Japan; and †Department of Life Science, Faculty of Science, Himeji Institute of Technology, Himeji 678-1297, Japan

Received May 1, 2000

Myosin V is an unconventional myosin thought tomove processively along actin filaments. To have hard
evidence for the high processivity, we sought to observe directly the movement by individual nativechick brain myosin V (BMV) molecules with fluorescent calmodulin. Single BMV molecules did exhibit
highly processive movement along actin filaments
fixed to a coverslip. BMV continued to move up to thebarbed end of its actin track, and did not readily detach from action. The barbed end, therefore, got
brighter with time, because of a constant stream of
BMV traffic. The maximum speed of the processive
movement was 1 ␮m/s, and the maximum actinactivated ATPase rate was 2.4 s ⴚ1. These values apparently imply that BMV travels a great distance, 400 nm,
per an ATPase cycle. © 2000 Academic Press
Key Words: motor protein; sliding movement; processivity; ATPase; actin; myosin.

The transport of vesicles and organelles to specificlocations is a fundamental process in cells. This process
is performed mainly by actin-based and microtubulebased protein motors. Of the unconventional myosinfamilies the class V myosins have been shown to transport synaptic and endoplasmic reticulum vesicles inneurons (1–3), pigment granules in melanocytes (4, 5),
and the vacuole in yeast (6, 7). Myosin V consists of two
N-terminal heads containing the actin-binding and
ATP hydrolysis sites, two extended neck domains eachof which contains six IQ motifs, and a tail domain
consisting of a coiled-coil region attached to a
C-terminal globular region (8 –10). The IQ motifs form
binding sites for calmodulin (CaM) and related lightchains (9 –11). Unlike myosin II, the tail does not selfassociate, and therefore myosin V does not form filaments (10, 12). The C-terminal globular region is believed to associate with cargo to be carried by myosin
To whom correspondence should be addressed. Fax: ⫹81 76-2645739. E-mail: tando@kenroku.kanazawa-u.ac.jp.
10006-291X/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.

V. The cycling rate of the actin-activated ATPase islimited by the ADP-release step, which allows myosin
V to spend a large part of the cycle time strongly boundto actin (13, 14). This kinetic nature has not been foundyet in the other myosin families.
Based on the cellular functions and the ATPase kinetics, myosin V has been inferred to be a processive motor
like the microtubule-based motors, kinesin (15, 16) anddynein (17), that is, a single myosin V molecule may beable to travel long distances along its actin track, repeating its ATPase cycles. The processivity is more directlysuggested by a recent observation that actin filaments
moved continuously over surfaces coated with BMV (myosin V purified from chick brain) at low densities, andswiveled about a single contact point as they moved (12).
Moreover, it was shown in an optical trapping assay thata single molecule of BMV pulled an actin filament successively for three-to-five, not many, steps before being
stalled by the optical trapping force (12). In the present
study we directly observed movement by individual BMV
molecules on fixed actin tracks, and found that BMV
travels over a distance of several micrometers without
detaching from actin, that is, BMV makes many steps insuccession.
MATERIALS AND METHODS
Proteins. BMV was prepared as described (18), stored at 0°C, andused within a week. Actin was prepared from rabbit skeletal muscle,
and purified as described (19). Bovine brain CaM and bovine serum
albumin (BSA; fraction V) was obtained from Sigma (USA). Streptavidine was obtained from Life Technologies (USA).
Cy3-labeling of calmodulin. After dialyzing against Buffer A (0.3
M KCl, 50 mM boric acid (pH 8.5), 0.1 mM CaCl 2), bovine brain CaM(15 ␮M) was mixed with 30 ␮M Cy3-Osu (Amersham Pharmacia
Biotech, UK) and incubated for 1 h at 25°C. After adding 5 mM
dithiothreithol (DTT) to stop the reaction, it was exhaustively dialyzed against Buffer B (25 mM KCl, 20 mM imidazole (pH 7.6), 2 mM
MgCl 2, 1 mM EGTA) plus 2 mM ␤-mercaptoethanol (␤-ME). The
concentration of Cy3 in the Cy3-CaM was estimated by the absorbance at 550 nm and the molar extinction coefficient, 1.5 ⫻ 10 5M ⫺1cm ⫺1.

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Preparation of Cy3-BMV. BMV (150 nM) was mixed with Cy3CaM (1.5 ␮M) in Buffer B plus 5 mM DTT, and incubated for 2 min
at 25°C. After adding CaCl 2 (final 1.01 mM), the mixture was incubated for 5 min at 25°C, followed by the addition of EGTA (final 4mM). To remove free Cy3-CaM, the sample was mixed with F-actin(final 1 ␮M), and centrifuged (148,000g, 1 h). The pellet was dissolved in Buffer C (250 mM KCl, 2 mM MgCl 2, 1 mM EGTA, 20 mM
imidazole (pH 7.6), 5 mM DTT, 2 mM ATP). The acto-Cy3-BMV wasapplied to a Q-Sepharose (Amersham Pharmacia Biotech, Sweden)column (volume, 400 ␮l). F-actin was removed from the sample bywashing the column with 20 ml of Buffer C. After washing thecolumn with 20 ml of Buffer C without ATP, Cy3-BMV was eluted
out with a linear gradient 0.25-1 M KCl with 20 mM imidazole (pH7.6), 2 mM MgCl 2, 1 mM EGTA and 5 mM DTT (total 30 ml), anddialyzed against Buffer B plus 2 mM ␤-ME.
In vitro actin-filament sliding assay. The assay was according toToyoshima et al. (20), with a slight modification (cover slips for flow
cells were treated with 0.2%, instead of 0.1%, nitrocellulose isopentyl
acetate solution for 1 min). F-actin was labeled with rhodaminegreen phalloidine (RGP). BMV or Cy3-BMV (100 nM, 20 ␮l) wasapplied to the flow cell (volume 10 –20 ␮l), and incubated for 2 min.
The cell was washed with 200 ␮l of Buffer B. Then, 50 ␮l of BSA (1mg/ml in Buffer B) was put into the cell, which was incubated for 2
min and was washed with Buffer B. RGP-labeled F-actin (8 nM, 100␮l) was applied to the cell and incubated for 2 min. Finally, the buffer
solution was replaced with motility buffer containing 20 mM imidazole (pH 7.6), 2 mM MgCl 2, 1 mM EGTA, 2 mM ␤-ME, 2 mM ATP,various concentrations of KCl, and an oxygen-scavenging system (0.2
mg/ml glucose oxidase, 0.36 mg/ml catalase and 4.5 mg/ml glucose).
Sliding movement of actin filaments was observed at 25°C using an
SIT video camera (C2741-08, Hamamatsu Photonics) connected to an
unmodified microscope (Olympus IX70; equipped with an oilimmersion objective, 100s, NA 1.35) with epifluorescence excitation
with a high-pressure mercury lamp and a filter set (U-MNIBA;
Olympus, Japan). The images were recorded with a Hi8 video cassette recorder (EVO-9659, Sony, Japan). The recorded images weredigitized with an image processor (Excel, Nippon Avionics, Japan)
and the two-dimensional coordinates of the rear end of each actin
filament were chased frame by frame. Length of a track connecting
these coordinates during a given period of time was calculated andaveraged over more than fifty actin filaments. The sliding velocity
was obtained by dividing the average value of the lengths of tracks
by the given period of time.
In vitro BMV movement assay. G-actin (10 ␮M) was mixed with
50 ␮M biotin-maleimide (Dojindo Laboratories, Japan) in a solution
containing 20 mM TES-HCl (pH 7.0), 0.2 mM CaCl 2, 0.1 mM NaN 3,
0.2 mM ATP, and incubated for 1 h at 25°C. After adding 5 mM DTT
to stop the reaction, the biotinylated G-actin was dialyzed against
the same buffer solution. The concentration of biotin incorporated
into G-actin was measured as described (19). One-to-one stoichiometric incorporation was achieved. The biotinylated G-actin was
copolymerized with nonlabeled G-actin (the molar ratio, 1:200) in
Buffer B. This ratio seemed important for observing processive
movement by Cy3-BMV. The resulting F-actin was labeled with RGPor phalloidine, and then fixed onto a coverslip as follows. First, BSA
was biotinylated with biotin-(AC 5) 2-Osu (Dojindo Laboratories, Japan) in a BSA/biotin molar ratio of 1:20 in 0.1 M boric acid, pH 8.8 for
90 min at 25°C, and dialyzed against Buffer B. The sample contained
2 biotins per BSA. The biotin-BSA (10 ␮M, 20 ␮l) was applied to aflow cell (volume 10 ␮l) made of two nitrocellulose-coated coverslips.
The cell was incubated for 2 min, and washed with 300 ␮l of Buffer
B. Streptavidin (0.5 mg/ml, 10 ␮l) dissolved in Buffer B was applied
to the cell, and incubated for 3 min. Unattached Streptavidin waswashed out with 600 ␮l of Buffer B. The partially biotinylated
F-actin (1 ␮M, 20 ␮l) was applied to the cell, and incubated for 2 min.
Unattached F-actin was washed out with 200 ␮l of Buffer B. Finally,
Cy3-BMV (1 nM) in 100 ␮l of motility buffers was applied to the cell.
Motility buffers are the same as those used for the actin-filament

FIG. 1. Distributions of spot intensities (arbitrary units) for (a)Cy3-CaM and (b) Cy3-BMV. The spot intensities were obtained byintegrating the intensity over a square (13 ⫻ 13 pixels) enclosing the
spot and subtracting a background integrated over the same square
after complete photobleaching of Cy3.

sliding assay. Fluorescence image of Cy3-BMV was observed using
an objective-type total internal reflection fluorescence (OTIRF) video
microscope, and recorded with a digital video cassette recorder (DSR20, Sony, Japan). The two-dimensional coordinates of each Cy3-BMV
were chased frame by frame. Length of a track connecting thesecoordinates during a given period of time was calculated and averaged over more than 100 Cy3-BMV molecules. The sliding velocity
was obtained by dividing the average value of the lengths of tracks
by the given period of time.
Microscopy. The OTIRF microscope was constructed as described(21), using an epifluorescence microscope (IX50; Olympus) equippedwith an oil-immersion objective lens (PlanApo 100⫻, NA 1.4) and arelay lens (PE5, 5⫻; Olympus) connected to an SIT video camera(C2400-08S; Hamamatsu Photonics). The excitation light source is a
diode green laser (model 4601-050, 532 nm, 50 mW; Uniphase)attenuated by two 20% neutral density filters.
ATPase assay. Actin-activated BMV and Cy3-BMV ATPase activities were measured at three different [KCl] with nine different
actin concentrations. The buffer solution contained 2 mM ATP, 20
mM imidazole (pH 7.6), 2 mM MgCl 2, 1 mM EGTA, and KCl (25, 100,or 200 mM). The concentration of BMV or Cy3-BMV was 30 – 60 nM.
Amounts of phosphate liberated at 25°C were quantified by themethod of Fiske and Subbarow (22). The ATPase activities were
determined by analyzing the amounts of phosphate liberated atseven time points by the least-squares criterion.
F-actin cosedimentation assays. BMV (150 nM) or Cy3-BMV (150nM) was mixed with 40 ␮M F-actin at 25°C in 2 mM ATP, 20 mM
imidazole (pH 7.6), 2 mM MgCl 2, 1 mM EGTA, 2 mM ␤-ME and threedifferent [KCl] (25 mM, 80 mM, or 150 mM). The samples (200 ␮l)were then immediately centrifuged (148,000g, 45 min, 25°C). The
supernatants were carefully aspirated and mixed with 50 ␮l SDS
sample buffer. The pellets were resuspended in the buffer solution(200 ␮l) plus 50 ␮l SDS sample buffer. Pellet and supernatant wererun on SDS polyacrylamide gels and silver-stained. As a control, it
was confirmed that BMV or Cy3-BMV alone was not sedimented,
irrespective of the presence of ATP.

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FIG. 2. Processive movement by individual BMV molecules along actin filaments. (a) Successive video images at 0.5-s intervals showing
processive movement by a single molecule of BMV along a fluorescent actin filament. The actin filament is labeled with RGP. A light spotin the left-hand side is moving toward the stationary light spot at the actin-filament end. KCl: 75 mM. Scale: 2 ␮m. (b) Successive video
images at 2-s intervals. The barbed end of the actin filament is getting brighter with time because of a constant stream of BMV traffic. A chainof light spots are visible on the actin filament labeled with nonfluorescent phalloidine. KCl: 25 mM. Scale: 2 ␮m. (c) A video image showing
bright spots formed 1 min after incubating BMV in the motility-assay cell. The actin filaments are labeled with nonfluorescent phalloidine.
KCl: 25 mM. Scale: 2 ␮m. The camera gain and the offset levels are different between (a), (b) and (c). In (b) and (c), these parameters forimaging are lower than those in (a). Therefore, we cannot compare the spot intensities in the three cases.

RESULTS AND DISCUSSION
To visualize BMV under the OTIRF microscope wefirst attempted to label BMV directly with amino-reactive
or SH-reactive fluorophores. The labeled BMV, however,
did not support in vitro actin-filament sliding. So, next,
we exploited the property of BMV that calmodulin (CaM)
attached to the neck region can dissociate partially in thepresence of a micromolar order of Ca2⫹, and the resulting
vacant CaM-binding sites can be refilled when [Ca2⫹] isreturned to a much lower level in the presence of excess
amounts of free CaM (10, 11). As the exogenous CaM, weused bovine CaM labeled with a fluorophore, Cy3 (Cy3/
CaM ⫽ 1.85). In this way a part of the endogenous CaM
was successfully replaced with the fluorescent CaM. This
sample of BMV contained 4.2 Cy3/BMV, indicating that
2.2 CaMs were replaced on the average. This Cy3-BMV
supported in vitro motility of actin filaments indistinguishably from that produced by non-labeled BMV (themaximum velocity is 0.55 ␮m/s). OTIRF video microscopy
was employed to observe Cy3-BMV molecules attached toa coverslip. We saw light spots on the monitor. To examine whether these spots represent individual Cy3-BMV
molecules, distribution of spot intensities of the Cy3BMV sample (IM) was compared with that of Cy3-CaM(I C) (Fig. 1). The ratio of the mean values, i.e., 具I M典/具I C典,

was 2.1. This value was quite similar to the average
number of substituted CaM measured in solution, indicating that Cy3-BMV is not oligomerized. Electron microscopic observation of Cy3-BMV also supported thisconclusion (data not shown).
We constructed an in vitro BMV motility assay system by attaching partly (0.5%) biotinylated actin filaments onto a streptavidine-coated coverslip. After attaching to an actin filament, Cy3-BMV moved
continuously and smoothly on its actin track (Fig. 2a).
At [KCl] lower than 100 mM, Cy3-BMV molecules often traveled up to the barbed ends of actin filaments.
After reaching the barbed ends, Cy3-BMV did notreadily detach from actin. The barbed ends were,
therefore, getting brighter with time because of a constant stream of Cy3-BMV traffic (Fig. 2b and 2c). At[KCl] higher than about 150 mM, Cy3-BMV often detached from actin before reaching the barbed end of its
actin track. The distance that Cy3-BMV traveled
seemed to fit single exponential statistics (Fig. 3a–3d),
and decreased with increasing [KCl] (Fig. 3e). The processive movement was still visible even at 400 mM
KCl, although the mean travel distance was small(about 660 nm). The rate of movement was 0.54 ␮m/s
at 25 mM KCl, increased moderately with increasing588

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FIG. 3. Processive movement by individual BMV molecules atvarious ionic strengths. (a– d) Distribution of distances traveled on
actin tracks by individual BMV molecules before detachment. Solid
curves: exponential fits with length constants, 2.39 ␮m (a), 1.76 ␮m(b), 1.17 ␮m (c), and 0.66 ␮m (d). The average travel distances areplotted as a function of [KCl] in (e). (f) The rate, as a function of[KCl], of actin-filament translation propelled by large ensembles of
BMV (closed squares), and the rate, as a function of [KCl], of processive movement by individual BMV molecules along their actin
tracks (closed circles).

[KCl], and reached a plateau (1 ␮m/s) at about 150 mM
KCl (Fig. 3f). This behavior is different from that ofkinesin. The rate of processive movement of kinesin is
independent of ionic strength (15).
The actin-activated BMV and Cy3-BMV ATPase activities were measured in 2 mM ATP and three different [KCl] with various concentrations of actin. The
results are summarized in Table 1. The ATPase activities displayed simple hyperbolic saturation behavior
as a function of [actin] (data not shown), i.e., V max/(1 ⫹K mA/[actin]). Irrespective of [KCl] both samples exhibited similar maximum turnover rates, V max (⬃2.4 s ⫺1).
They also showed similar K mA. These values agreed withthose reported previously for native BMV (23). These

results, together with the same rate of actin translocation propelled by BMV and Cy3-BMV, guarantee that
the CaM-exchange does not alter the properties ofBMV. The rate of movement (0.54 ␮m/s at 25 mM KCl,
1 ␮m/s at [KCl] higher than 150 mM) of single Cy3BMV molecules and the ATPase turnover rate (2.4 s ⫺1)
suggest that Cy3-BMV may travel a great distance(230 – 400 nm) per an ATPase cycle, although it does
not prove. To accommodate the maximum rate of movement (1 ␮m/s) with the step size (ca. 36 nm) observedby an optical trapping assay with native BMV in theabsence of Ca 2⫹ and in various [ATP] (12), we may beable to consider two hypothetical cases. Case 1: The 36
nm step is not directly coupled with each ATPase cycle.
Within one ATPase cycle, BMV could make multiple(6 –11) 36 nm-step advances in succession, as has beensuggested with skeletal muscle myosin subfragment-1(24). This view may be supported by an observationthat dwell durations between 36 nm-steps were much
shorter than the ATPase cycle time (12). If so, the dwell
durations should be insensitive to [ATP], as long as‘rigor head’ formed in low [ATP] does not hinder its
nucleotide-bound partner from repeating 36 nm-steps
in succession. However, observed dwell durations increased with decreasing [ATP] (12). Case 2: A large
fraction (more than 90%) of BMV may be in an inhibited or low-activity state where it does not interact
with actin, and therefore BMV not in such a state may
have much higher ATPase activity (14 s ⫺1/head, or 28s ⫺1/BMV), 12 times larger than the value measured
here. If so, it is understandable that large ensembles of
BMV molecules produced actin-filament sliding at a
rate half that with movement of single BMV molecules.
We tested this hypothesis by cosedimentation assays
for BMV and Cy3-BMV binding to F-actin in 2 mM
ATP. It came out that this hypothesis did not fit, because a large fraction of BMV and Cy3-BMV were
cosedimented with F-actin, even at 150 mM KCl (Fig.
4). This cosedimentation does not mean that our BMV
sample contains a large fraction of denatured BMV,
but that BMV is mainly in the strong-binding states
during the ATPase cycle (13, 14). If denatured, it must
produce resistive drag force against actin-filament sliding, as has been observed with skeletal heavy meromyTABLE 1

Steady-State ATPase Rate of Acto-BMV and Acto-Cy3-BMV
at Three Different KCl Concentrations
V max (s ⫺1)

K mA (␮M)

KCl
(mM)

BMV

Cy3-BMV

BMV

Cy3-BMV

25
100
200

2.37
2.20
2.47

2.41
2.36
2.42

0.39
1.10
7.09

0.41
1.28
7.00

Note. V max, the maximum ATPase rate; K mA, actin concentration
where the ATPase rate is half of V max.

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FIG. 4. Greyscale ccd images of silver-stained SDS-gels from F-actin cosedimentation assays. Left panel, BMV (150 nM), and right panel,
Cy3-BMV (150 nM), were incubated with 40 ␮M F-actin in the presence of 2 mM ATP and three different [KCl] (25 mM, 80 mM, 150 mM).
Pell, 20 ␮l sample of pellets resuspended in the original volume of the buffer solution was loaded. Sup, 20 ␮l sample of supernatants, were
loaded. The silver-staining conditions are slightly different between left and right panels.

osin, wherein only 15–20% denatured fraction of heavy
meromyosin is sufficient to stop actin-filament sliding.
The ATPase activity as high as 14 s ⫺1/head has beenreported for native BMV in the presence of a micromolar order of Ca 2⫹ (23) and for truncated, single-headed
myosin V constructs with one or two single light chainsin the absence of Ca 2⫹ (13, 25). The high ATPase activities are, however, ascribed to the absence of an inhibitory effect of CaM on the acto-BMV ATPase reaction(23). Without the inhibitory effect of CaM, BMV does
not support in vitro motility of actin filaments (10).
Here, we provided hard evidence that BMV is ahighly processive motor that can travel over a distanceof many micrometers without detaching from actin.
However, it raised a new riddle about the mechanochemical coupling of this protein motor, which should
be solved in the future.
ACKNOWLEDGMENTS
We thank K. Saito for the OTIRF microscope. This work was
supported partly by the Ministry of Education, Science, Sports andCulture of Japan (Grant-in-Aid for Scientific Research on Priority
Areas (A) to T.A.) and by the Sasagawa Scientific Research Grant
from The Japan Science Society to T.S.

REFERENCES
1. Prekeris, R., and Terrian, D. M. (1997) J. Cell Biol. 137, 1589 –1601.
2. Evans, L. L., Lee, A. J., Bridgman, P. C., and Mooseker, M. S.
(1998) J. Cell Sci. 111, 2055–2066.
3. Tabb, S., Molyneaux, B. J., Cohen, D. L., Kuznetsov, S. A., andLangford, G. M. (1998) J. Cell Sci. 111, 3221–3234.
4. Provance, D. W., Wei, M., Jr., Ipi, V., and Mercer, J. A. (1996)
Proc. Natl. Acad. Sci. USA 93, 14554 –14558.
5. Wu, X. F., Bowers, B., Wei, Q., Kocher, B., and Hammer, J. A.
(1997) J. Cell Sci. 110, 847– 859.
6. Hill, K. L., Catlett, N. L., and Weisman, L. S. (1996) J. Cell Biol.
135, 1535–1549.

7. Catlett, N. L., and Weisman, L. S. (1998) Proc. Natl. Acad. Sci.
USA 95, 14799 –14804.
8. Espindola, F. S., Esprefico, E. M., Vcoelho, M., Martins, A. R.,
Casta, F. R. C., Mooseker, M. S., and Larson, R. E. (1992) J. Cell
Biol. 118, 359 –368.
9. Esperafico, E. M., Cheney, R. E., Matteoli, M., Nascimento,
A. A. C., De Camilli, P. V., Larson, R. E., and Mooseker, M. S.
(1992) J. Cell Biol. 119, 1541–1557.
10. Cheney, R. E., O’shea, M. K., Heuser, J. E., Coelho, M. W.,
Wolonski, J. S., and Espreafico, E. M. Cell 75, 13–23.
11. Cameron, L. C., Carvalho, R. N., Araujo, J. R. V., Santos, A. C.,
Tauhata, S. B., Larson, R. E., and Sorenson, M. M. (1998) Arch.
Biochem. Biophys. 355, 35– 42, doi:10.1006/abbi.1998.0700.
12. Mehta, A. D., Rock, R. S., Rief, M., Spudich, J. A., Mooseker,
M. S., and Cheney, R. E. (1999) Nature (London) 400, 590 –593.
13. De La Cruz, E. M., Wells, A. L., Rosenfeld, S. S., Ostap, E. M.,
and Sweeney, H. L. (1999) Proc. Natl. Acad. Sci. USA 96, 13726 –
13731.
14. Titus, M. A. (1997) Curr. Biol. 7, R301–R304.
15. Vale, R. D., Funatu, T., Pierce, D. W., Romberg, L., Harada, Y.,
and Yanagida, T. (1996) Nature (London) 380, 451– 453.
16. Howard, J., Hudspeth, A. J., and Vale, R. D. (1989) Nature(London) 342, 154 –158.
17. Sakakibara, H., Kojma, H., Sakai, Y., Katayama, E., and Oiwa,
K. (1999) Nature (London) 400, 586 –590.
18. Cheney, R. E. (1998) Methods Enzymol. 298, 3–18.
19. Kunioka, Y., and Ando, T. (1996) J. Biochem. 119, 1024 –1032.
20. Toyoshima, Y. Y., Kron, S. J., McNally, E. M., Niebling, K. R.,
Toyoshima, C., and Spudich, J. A. (1987) Nature (London) 328,
536 –539.
21. Tokunaga, M., Kitamura, K., Saito, K., Iwane, A. H., andYanagida, T. (1997) Biochem. Biophys. Res. Commun. 235, 47–
53, doi:10.1006/bbrc.1997.6732.
22. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem. 14, 1–10.
23. Nascimento, A. A. C., Cheney, R. E., Tauhata, S. B. F., Larson,
R. E., and Mooseker, M. S. (1996) J. Biol. Chem. 271, 17561–
17569.
24. Kitamura, K., Tokunaga, M., Iwane, A. H., and Yanagida, T.
(1999) Nature (London) 397, 129 –134.
25. Trybus, K. M., Krementsova, E., and Freyzon, Y. (1999) J. Biol.
Chem. 274, 27448 –27456.

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