A M . ZOOLOGIST, 7:457-464 (1967).
ATP-Driven Oscillation of Glycerol-Extracted Insect Fibrillar Muscle:
Mechano-Chemical Coupling
JOH. CASPAR RUEGG
Department of Physiology, Max-Planck Institute for Medical Research,
Heidelberg, and Department of Cell-Physiology, Ruhr University,
Bochum, Germany
SYNOPSIS. Synchronous (fibrillar) insect flight muscle oscillates according to a
myogenic rhythm. The oscillator is built into the contractile structure, which can
oscillate and perform work in a constant chemical environment with ATP as the
only source of energy, when it has been isolated by glycerol-extraction. During oscillation, changes in tension follow changes in length with a delay, since contractile
activity is switched on and off with a delay by elongation and shortening of the
glycerol-extracted fibers (stretch activates, and release of the fibers deactivates, the
contractile ATPase). Consequently, sinusoidal stretch and release induce oscillation
(driven oscillation) associated with extra ATPase activity. The latter is proportional
to the power-output, implying a biochemical Fenn-effect. Power-output and ATPase
activity can be increased by raising the concentration of calcium or—at constant
chemical conditions—by increasing the frequency or the amplitude of driven oscillation, demonstrating a mechano-chemical coupling between mechanical performance
(product of delayed tension and speed of shortening) and enzymatic activity.
OSCILLATION OF INSECT
FIBRILLAR FLIGHT MUSCLE
In vertebrate skeletal muscle and presumably also in synchronous non-fibrillar
insect flight muscle contractile activity is
switched on by an increase and switched
off by a decrease of the intracellular calcium ion concentration. The latter is
regulated by calcium release from and
by uptake by the sarcoplasmic reticulum
(for survey, see Hasselbach, 1964). In insect
fibrillar muscle the sarcoplasmic reticulum
is poorly developed (Smith, 1961) and there
is evidence that activity is not only regulated by calcium ions but also by a different mechanism. In situ or after attachment to an elastic auxotonic recording
lever system these muscles contract and
relax rhythmically when stimulated. The
frequency of this oscillation is independent of the strength and frequency of electrical stimulation and it is not associated
with action potentials in synchrony with
the oscillation (Pringle, 1949, 1954; Darwin and Pringle, 1959; Machin and Pringle, 1959). While these experiments indicate that the movement is apparently not
controlled by an excitable membrane, ex-
periments with glycerol-extracted single
dorso-longitudinal muscle fibers of the
waterbugs, Lethocerus maximus and Lethocerus annulipes, demonstrate that the oscillatory movement is not only independent
of a functional cell membrane but also
of the sarcoplasmic reticulum (Jewell and
Riiegg, 1966). Such fibers represent a functionally isolated myofibrillar system. When
attached to an elastic auxotonic lever system they oscillate after immersion in an
activating ATP solution for up to 23 hr,
at an amplitude of up to 1.5% of the
fiber length and at a frequency which is
near the resonance frequency of the lever
system (e.g., about 20 c/sec). The activating solution contains Mg-ATP in a concentration of 5 mM/1 which is sufficient
to maintain an ATP concentration of
about 2-3 mM in the center of the fiber
(see Mannherz, 1966). The pH is buffered
at 6.5 or 7.0 with histidine, and the calcium concentration is adjusted to 10~710- 6 M and buffered with calcium-EGTA
(Ethyleneglycol-bis (/3-Amino-ethyl-ether) N,
N'-tetra acetate) according to Portzehl, et
al. (1964). The amplitude, but not the
frequency, of oscillation depends on the
calcium ion concentration. Oscillation
(457)
458
JOH. CASPAR RUEGG
stops when stimulation ends: in living
muscle after cessation of electrical stimulation, and in glycerol-extracted fibers after
immersing the fiber in a relaxing ATP solution in which sufficient EGTA has been
added to reduce the free calcium concentration to 10~8 M or less; azide is added
to inhibit mitochondrial activity. Since the
functionally isolated myofibrillar system,
consisting essentially of actin and myosin
filaments, contracts and relaxes during oscillation up to a million times with ATP
as the only energy source and at a constant (i.e., buffered) level of ionized calcium, it seems that in the living cell, too,
oscillation is ATP-driven and dependent
on the presence of calcium ions but independent of the rapid "give and take" of
calcium ions by the sarcoplasmic reticulum (SR). Thus, during flight, little energy is wasted for the calcium-pumping
action of the SR.
The oscillatory automatism functions
only if the fibers are attached to an auxotonic lever system of suitable natural frequency. If on the other hand the extracted
fibers are attached to an isometric lever
system they do not oscillate but develop
tension which at pH 7 is approximately
proportional to the logarithm of the calcium ion concentration. The calcium concentration for half-activation of myofibrillar ATPase activity (v. Brocke, 1966) and
development of tension is about 10~7 M
at pH 7.0 but much higher at more acid
pH (Schadler, 1966).
by the 'built-in oscillator'. In an activating
solution the fiber shortens, thereby lowering the contractile activity so that the
fiber relaxes and lengthens. The lengthening occurs because of the restoring force
of the elastic lever which stretches the fiber;
it evokes a counterforce of increased muscular activity associated with increased release of chemical energy from ATP. Consequently, the system shortens again. Such
a system would tend to oppose any change
in length by a negative feedback mechanism. In order to oscillate, supposedly the
contractile counterforce must be evoked
with a delay after the change in length,
so that lengthening is followed by a delayed
rise in tension and shortening by a delayed
fall in tension. Under these conditions
the fiber puts out the power necessary to
overcome the damping of the lever system
DLM FIBREBUNDLE IN ATP-SALTSOLUTION
3 I0'7'M Ca2'; pH 6 5 • 23'C
ACTIVATION BY STRETCH
1 Smg
TOTAL TENSION
5
to
o
1
PASSIVE TENSION
•2.5
REGULATION OF OSCILLATORY MOVEMENT
During oscillation, tension and length
change rhythmically, and since this is possible at constant calcium concentration in
an activating ATP solution it is required
that a 'built-in' oscillator switches the
contractile activity on and off at the rhythm
of oscillation. Since this happens under
auxotonic but not under isometric recording conditions, with a rhythm which is
always near the natural frequency of the
chosen lever system, activity is—as suggested
by Pringle (1965)—obviously controlled by
the muscle fiber length which is sensed
+5
'75% L.
LENGTH
3 tO'7M Co1'
10 MIN.
W7.5'U L.
FIG. 1. Top: Increase of total tension, passive tension, and myofibrillar ATPase activity in an activating solution of ATP after stretching extracted
fiber bundles by different amounts. Below: Contraction in activating ATP solution before and after
stretching the extracted fibers in a calcium-free,
relaxing "solution.
4S0
OSCILLATION OF INSECT FIBRILLAR MUSCLE
LETHOCERUS
DLM
GLYCERINATED SINGLE FIBRE
70u THICK. aScm LONG• SINUSOIDAL STRETCH
FREOENCY'2/SEC.
AMPLITUDE •?£%/..
W6M
OS SEC
Co2'
0.5 SEC
SOmg
TENSION
pH 65
SmM ATP-Mg
70mM KCI
lOmM HIS.
30 mg
LENGTH
SOmi
TENSION
TENSION
LENGTH
0.1mm '
LENGTH 0.1mm '
FIG. 2. Driven oscillation o£ glycerol-extracted
fibers: left, in relaxing solution; right, in activating
ATP solution. Top: oscillation of tension and
length. Below: the tension-length diagram. The
area enclosed by the loop is the work done by the
fibers in each cycle o£ oscillation when the fiber
is driven t oscillate in an activating ATP solution.
Maximal power-output ~ 1 p. cal/cm/fiber/min.
immersed in the bath, and maintains
oscillation.
The increase in contractile activity and
ATPase activity (Riiegg and Tregear, 1966)
by forcible elongation above resting length
is shown in Fig. 1. The effect is reversible
and it happens in the activating ATP
solution regardless of whether the extension is carried out in that solution or in
a calcium-free relaxing solution. In the
latter case the stretch evokes a small increase in passive tension (Fig. 1) on which
much larger tension is superimposed when
calcium ions are added. The activation of
extra tension is proportional to the extra
ATPase activity produced by stretching
and it shows the same dependence on calcium ion concentration as the actomyosin
ATPase. The concentration needed for
half-activation and presumably (see A.
Weber, et al., 1963) for half-saturation of
the available calcium binding sites does
not change much after activating the prep
aration by stretch. Thus, it is unlikely
that the stretch-activation responsible for
the delayed rise of tension during oscillation is caused by a stretch-induced increase in affinity for calcium ions.
The delay in the rise of tension after
stretching is clearly seen after abrupt
stretches (Jewell and Riiegg, 1966) or when
the preparation is forcibly stretched and
released in a sinusoidal fashion while immersed in an activating ATP solution (Fig.
2)During sinusoidal stretch and release, the
tension reaches its maximal value after
the maximal degree of stretch, i.e., during
the release and at any length of the oscillation cycle the tension is larger during
the release half-cycle than during the
stretch half-cycle so that the tension-length
diagram has an ellipse-like shape which is
described in a counterclockwise fashion.
Clearly, during this driven oscillation the
fibers put out power while oscillating so
that the energy required to stretch the
preparation is less than the energy liberated on the release. The driven oscillation
experiment demonstrated that it is the
460
JOH. CASPAR RUEGG
changes in length which are sensed by the
"built-in" oscillator and which switch the
contractile activity on and off so that the
fibers oscillate actively and put out power
which in free oscillation experiments is
necessary to overcome the damping of the
system.
POWER-OUTPUT
If the change of delayed tension in the
driven oscillation experiments is measured
in "dynes" and the amplitude of the lengthchange in "cm" the area enclosed by the
tension-length diagram measures in "ergs"
the work done by the preparation in each
cycle of driven oscillation.
During sinusoidal stretch and release,
active tension and a delay between
changes in tension and length are only
observed when the ATP-saturated fibers are
activated with calcium ions. In a Ca2+"free" relaxing medium ([Ca2+] less than
10- 8 M) the sinusoidally stretched preparation is not stretch-activated and it behaves like an elastic body, i.e., there are
hardly any tension-lagging length-changes
and no work is either produced or absorbed. Absorption of work does occur
during ATP-starvation of the fibers when
length-changes lag behind tension-changes,
indicating a positive viscous modulus.
Compared with the free oscillation method the driven oscillation method has the
advantage that the frequency can be easily
varied and selected to give a maximal work
output per cycle of oscillation (Fig. 3, top)
or maximal power-output (see Jewell
and Riiegg, 1966). Since the power-output
is proportional to the delayed changes in
tension (quadrature component of tension)
and also to the velocity of movement, it
is possible to increase the power in a given
activating ATP solution by increasing
either the frequency or the amplitude of
oscillation. Increasing the amplitude at a
given frequency of oscillation, however, not
only increases the speed but also the degree
of stretch-activated, delayed tension, so that
apparently the power-output increases in
proportion to the square of the amplitude
of oscillation. The maximal value obtained
in these experiments was about 1.0 /x
cal/cm fiber per min at a frequency of
2.5 c/sec (Fig. 3, below).
ENERGETICS: RELATION OF
POWER-OUTPUT AND ATPASE ACTIVITY
During oscillation the single fibers or
small bundles of fibers were suspended in
Perspex baths containing 0.3 ml activating
ATP solution; after poisoning of the mitochondrial ATPase with azide, the ATPase
activity of the fibers, and thus the rate of
total energy release, could be measured by
estimating the phosphate liberated (Riiegg
and Tregear, 1966; Marsh, 1959). It was
then possible to test whether the functionally isolated contractile machinery, like
vertebrate muscle, liberated energy at a
higher rate when it produces power by
EFFECT OF OSCILLATIONFREOUENCY ON POWER % OUTPUT
{FROM JEWELL AND RUEGG)
%
<)-•>- „
100\
0\
POWER
SO'
10
15
20 C/SEC.
EFFECT OF AMPLITUDE OF OSCILLATION.
ON POWER OUTPUT WLM-FIBREBUNDLES IN ATPSALTSOLUTION
•>! OSC 2.S-3C/SEC.
AMPLITUDE]1
3
a •
o
;
2
3V, L.
LENGTH CHANGE {.AMPLITUDE)
FIG. 3. Driven oscillation of extracted fiber bundles.
Top: the effect of the frequency of oscillation.
Below: the effect of varying the amplitude of oscillation; each fiber bundle (all from the same
muscle) was driven both at about 1.5% the muscle
amplitude and at about 2.3%. Crosses = means;
these are replotted against the square of the
amplitude (broken line). Conditions as in Figure 4.
OSCILLATION OF INSECT FIBRILLAR MUSCLE
EFFECT OF OSCILLATION AMPLITUDE ON EXTRA ATP-ASE
AND POWER
.
o
Q
033 FIBRE BUNDLES
§ ATP SALTSOLUTION
S 3W' M Ca"
g
6 BUNDLES
k (OF 2-5 FIBRES)
a
2 -3c /SEC
I,," 3IO~'M Ca"
% pH 6 5, 2CTC
COL?
02'
/
2
3'AL.
AMPLITUDE OF OSCILLATION
03
9 02
r.08S
b~ 3000 CAL/MOL ATP
POWER
uCALIMIN./cm FIBRE
OS
W
FIG. 4. Driven oscillation of extracted fiber bundles. Above: the effect of the amplitude of oscillation on the extra ATPase activity induced by oscillation. Each fiber bundle (all from the same
muscle) was driven at about 1.5% muscle-length
amplitude and at about 2.3%. For comparison,
compare the effect of amplitude on the power
average (squares with broken line). Below: the
extra-ATPase activity replotted against poweroutput. The extra ATPase activity is the difference between the total ATPase activity during
oscillation about a mean length and the ATPase
activity of the preparation kept isometrically contracted at the mean length and in the same medium [5 mM Mg-ATP, 30 mM KC1, 20 mM histidine-buffer, pH 6.5, 4 mM Ca-EGTA calcium
buffer (ionized calcium, 3 X 10-' M) 10 mM azide,
20°C].
shortening against tension than during
isometric contraction. In vertebrate muscle
the liberation of extra energy is proportional to the work done (Fenn) and this
can also be demonstrated biochemically in
living muscle (Davids, 1965; Marelchal,
1964; Carlson, et al, 1963). These studies
with living muscle however do not show
which part of the cell adapts the energy
output to the amount of work done. The
following experiments show that at least in
461
insect muscle the adaptation takes place in
the contractile structure, since the ATPase
activity is much larger when the muscle is
allowed to shorten against a load and perform oscillatory work, than (under the
same ionic conditions) during isometric
contraction at the mean length or even at
the maximal length of the fiber. The activation of the ATPase activity is proportional to the rate of doing work. This is
shown by Figure 4 for experiments with
fiber bundles of Lethocerus annulipes
which were sinusoidally stretched and
released at a frequency of about 2-3 c/sec,
first at a mean amplitude of about 1.5% of
the rest-length, and then at an amplitude
of about 2.3% of the rest-length. The
mechano-chemical coefficient relating work
and consumption of ATP is about 3,000
cal per mol of ATP split. Note that both
the power-output and the ATPase activity
increase in proportion to the square of
the amplitude. Therefore, both power and
extra ATPase activity appear to be proportional to the delayed tension and to the
length or velocity of shortening (or stretch)
during each cycle of oscillation.
At constant speed or length of shortening the amount of delayed tension and the
power-output can be increased by increasing the concentration of Ca2+. From
about 10~ 7 -l0- 6 M the increase of delayed
tension and oscillatory extra ATPase activity is almost proportional to the logarithm of the calcium concentration (Fig.
5); above 10- 8 M, however, the extra
ATPase activity produced on oscillation
increases further while the power-output
decreases. This apparent uncoupling of
ATPase activity and power-output associated with a decrease in the mechanochemical coefficient is characteristic for
the "high tension state" described by Jewell
and Riiegg (1966) which is a diffusional
artifact. At high calcium concentrations
and/or at high amplitudes of oscillation,
ATPase activity is so much increased that
the core of the fibers, or of the fiber bundle,
is no longer supplied with ATP. The ATPdeprived core does not split ATP, and because of its visco-elastic properties it absorbs
JOH. CASPAR RUEGG
462
ACTIVATION BY £a'3 AND STRETCH
OF ATP-ASE ACTIVITY
AND ACTIVE TENSION
mg
pH 6.5, 22'
. - - " } % STRETCH
DLM-FIBRES
DMFBR
n
, ' ' (6mj MISSIVE TEN
3
SION)
^
J% STRETCH
(Smg PASSIVE
TENSION)
m
Ca1* EFFECT ON DELAYED TENSIONOSCILLATORY
POWER AND EXTRA ATP-ASE ACTIVITY
wo
EXTRA STRETCH
ATP}ASE
SO
/ ' / /
/
' OSCILLATORY
OSCILLATORY
EX1RA~ATP~AS~E
\
FIG. 5. The dependence of myofibrillar ATPase
activity and oscillatory extra ATPase activity on
calcium ions. Also shown: power-output, tension,
stretch-induced ATPase and tension o£ fibers.
work produced by the ATP-splitting, oscillating myofibrils.
Diffusional studies of Mannherz (1966)
have shown that in non-oscillating fibers
and at fairly low calcium concentration the
ATP gradient between the outside and the
center of the fiber is about 2 raM but increases to 4-5 niM when the ATPase activity increases during oscillation. This
means that a concentration of ATP of 5
mM is just sufficient to supply the whole
fiber with ATP during oscillation and
power-output at low calcium concentration,
but that it is not sufficient to do so at the
very high ATPase activities produced by
high calcium concentration and/or high
amplitudes of sinusoidal stretch. Since the
ATP gradient increases during oscillation
about in proportion to the work done and
to the ATPase activity, it would seem
to follow that the apparent diffusional
coefficient changes little on oscillation.
MECHANO-CHEMICAL COUPLING
ATPase activity in a given ionic medium appears to be coupled to the mechanical performance of the preparation, on
the one hand to the degree of stretch and
the extra tension produced on stretch and
on the other hand to the power-output
during oscillation. Since the power-output
and the oscillatory extra ATPase are proportional to both the extra delayed tension
(induced by the oscillatory stretch) and to
the velocity of movement (either shortening or stretch) it would seem that oscillatory extra ATPase activity is coupled to extra tension and to speed of movement. The
extra ATPase of oscillation shows the same
dependence on the calcium concentration
as the extra ATPase produced by stretch
and the actomyosin ATPase (Fig. 5).
Thus, it follows that it is an actomyosin
ATPase and requires the interaction of
actin with myosin for the splitting of ATP.
There are about 1.3 p moles of crossbridges in 1 cm fiber of the dorso-longitudinal muscle of Lethocerus cordofanus
(Chaplain and Tregear, 1966). If the crossbridge content is similar in fibers of
Lethocerus maximus (see Reedy, Holmes,
and Tregear, 1965) they will split about 3
ATP molecules per cross-bridge in each
cycle of oscillation (i.e., 4-6 p mol/cm
fiber/minute/cycle during maximal oscillation at maximal amplitude (3-4% Lo);
with Lethocerus annulipes fiber the values
were less (see Fig. 3). This could mean
that during each cycle cross-bridges act
only once and liberate one ATP molecule per myosin molecule (assuming 3
myosin molecules per cross-bridge (see
Chaplain and Tregear). Alternatively it
may be assumed that 1 ATP molecule is
split per cross-bridge action but that each
cross-bridge acts several times during an
oscillation cycle at large amplitude. By
analogy with vertebrate muscle, it seems
likely, then, that this repetitive and
sequential action takes place during
shortening rather than during lengthening,
i.e., in that part of the oscillatory cycle
where delayed extra tension is generated.
One may speculate that during oscil-
OSCILLATION OF INSECT FIBRILLAR MUSCLE
lation, delayed extra tension is proportional to the number of reactive myosin
cross-bridges interacting with actin during
the shortening phase and each of them
splitting ATP at a rate which is proportional to the rate at which actin sites move
past and react with these myosin bridges
(i.e., the speed of shortening). In this case
the extra splitting of ATP during shortening would occur at a rate which is proportional to the product of the number of
reactive cross-bridges (which is related to
the amount of delayed tension and depends
on the extent of stretch and of the calcium
concentration) and of the speed of shortening. In other words it is proportional to
power-output.
However, if the speed of movement is
too high (Riiegg and Tregear, 1966) the
ATPase activity and power decrease again
because there is not sufficient time
for the interaction to take place and to
produce delayed tension. In this working
hypothesis the contraction phase of the
oscillating muscle may be comparable to
contraction of vertebrate muscle where the
rate of shortening is related to the rate of
release of energy from ATP in a sliding
system of actin and myosin filaments. The
nature of the energy-transfer in this molecular machine is still unknown but the use
of functionally isolated, contractile, actomyosin machinery (where output of energy
and consumption of ATP can be measured at the same time) would seem to offer
a promising approach.
SUMMARY
During insect flight, cross-striated fibrillar flight muscles contract and relax
at a rate which is determined by the natural frequency of the thorax-wing system
and which in certain small insects amounts
to as much as 1000/sec. The oscillatory contractile mechanism of these muscles is an
automatized molecular machine of actin
and myosin filaments which can be functionally isolated by glycerol-extraction of
the muscle fibers. After isolation it can be
driven by adenosinetriphosphate in the
presence of Mg and Ca-ions to give oscilla-
463
tory power-output of up to 20 gcm/sec/g
muscle with an efficiency of up to 3000 cal
per mol of ATP split. The contraction and
relaxation of extracted fibers occur at a
constant calcium concentration with a frequency near to the resonance frequency of
the lever system suggesting that in the
cell the oscillatory movement is not controlled by the uptake and release of calcium
but by a built-in length-sensing myofibrillar automatism which is activated by extension and deactivated by shortening with
a delay.
Under a variety of chemical and mechanical conditions, the ATPase activity, or
the rate of energy-release, is automatically
adapted to the power-output to which it is
proportional. The nature of the mechanochemical coupling between ATPase activity and the degree of passive stretchtension and/or power-output are discussed
in terms of a model of sliding filaments.
REFERENCES
Boettiger, E. G. 1957. The machinery of insect
flight. In B. T. Scheer, (ed.), Recent advances
in invertebrate physiology. Univ. of Oregon Publ.
p. 117-142.
v. Brocke, H. H., and J. C. Riiegg. 1965. Fraktionierung der ATPase fibrillarer Insektenmuskulatur.
Helv. Physiol. Acta 23, C 79-C 81.
v. Brocke, H. H. 1966. The activating effects of
calcium ions on the contractile systems of insect
fibrillar flight muscle. Pfliiger's Archiv. 290:70-79.
Chaplain, R. A., and R. T. Tregear. 1966. The
mass of myosin per cross-bridge in insect fibrillar
flight muscle. J. Mol. Biol. 21:275-280.
Davies, R. E. 1965. The role of ATP in muscle
contraction. In W. M. Paul, E. E. Daniel, C. M.
Kay, and G. Monckton, (ed.), Muscle. Pergamon,
New York.
Hasselbach, W. 1964. Relaxing factor and the relaxation of muscle. Progr. Biophys. 14:167-222.
Jewell, B. R., and J. C. Riiegg. 1966. Oscillatory
contraction of insect fibrillar muscle after glycerol
extraction. Proc. Roy. Soc. (London), B., 164:
428-459.
Machin, K. E., and J. W. S. Pringle. 1959. The
physiology of insect fibrillar muscle. II. Mechanical properties of a beetle flight muscle. Proc.
Roy. Soc. (London-), B, 151:204-225.
Machin, K. E., and J. W. S. Pringle, 1960. The
physiology of insect fibrillar muscle. III. The
effect of sinusoidal changes of length on a
beetle flight muscle. Proc. Roy. Soc. (London),
B, 152:311-330.
Mannherz, H. 1966. ATP-Spalting und Diffusion
464
J O H . CASPAR RUEGG
in oscillierenden extrahierten Muskelfasern. Pfliiger's Archiv. 291:94.
Marechal, G. 1964. Le metabolisme de la phosphorylcreatine et de l'adenosine triphosphate durant la contraction musculaire. Ed. Arscia. Bruxelles.
Marsh, B. B. 1959. The estimation of an inorganic
phosphate in the presence o£ adenosine triphophate. Biochim. Biophys. Acta 32:357-361.
Portzehl, H., P. C. Caldwell, and J. C. Riiegg. 1964.
The dependence of contraction and relaxation
of muscle fibers from the crab, Maia squinado,
on the internal concentration ot free calcium
ions. Biochem. Biophys. Acta 79:381-591.
Pringle, J. W. S. 1949. The excitation and contraction of the flight muscles of insects. J. Physiol.
108:226-232.
Pringle, J. W. S. 1954. The mechanisms of the myogenic rhythm of certain insect striated muscles.
J. Physiol. 124:169-291.
Pringle, J. W. S. 1965. Locomotion: flight, p. 283329. In The physiology of insecta. Vol. 2. Academic Press, New York.
Reedy, M. K., K. C. Holmes, and R. T. Tregear.
1965. Induced changes in orientation of the
cross-bridges of glycerinated insect flight muscle.
Nature. 207:1276-1280.
Ruegg, J. C, and R. T. Tregear. 1966. Mechanical
factors affecting the ATPase activity of glycerolextracted insect fibrillar flight muscle. Proc. Roy.
Soc. (London), B, 165:497-512.
Schadler, M. H. 1966. Calciumaktivierung von Spannung und ATPase contraktiler Systeme verschiedener Muskelarten. Pfliiger's Archiv 291:93.
Smith, D. S. 1961. The structure of insect fibrillar
flight muscle. A study made with special reference
to the membrane systems of the fiber. J. Biophys.
Biochem. Cytol. 10:123-158.
Weber, A., and R. Herz. 1963. The binding of
calcium of actomyosin systems in relation to their
biological activity. J. Biol. Cliem. 238:599-605.