J. comp:Physiol.
129, 163-167
(1979)
Journal
of Comparative
Physiology.
B
(Q by Springer-Verlag
1979
Calcium Regulatory Proteins and Temperature Acclimation
of Actomyosin A TPase from a Eurythermal Teleost (Carassius auratus L.:
Ian
A. Johnston
Department
of Physiology,
.
University
of St. Andrews, St. Andrews, Fife, Scotland
Accepted September 19, 1978
Summary. Goldfish (Carassius auratus) were acclimated for 5 months at temperatures of either 2 °C
or 31 °C. Natural actomyosin was prepared from
white myotomal muscle and its Mg2+Ca2+ ATPase
activity determined. Temperature acclimation results
in adaptations in substrate turnover number and
thermodynamic activation parameters of the A TPase.
When assayed at 31 °C the Mg2+Ca2+ ATPase of
natural actomyosin was 4 times higher in 31 °C than
2.°C acclimated fish. Arrhenius plots of natural actomyosin A TPase from cold acclimated fish show a
break in slope at 15-18 °C. In contrast, the temperature dependence of warm acclimated actomyosin was
linear. Activation enthalpy (L1H*) of the ATPase,
calculated over the range 0-16 °C, was approximately
8,000 cal/mole lower in 2 °C than 32 °C acclimated
fish.
In contrast, desensitised actomyosins from which
the calcium regulatory proteins have been removed
show a linear temperature dependence in the range
0-32 °C and have similar properties in 2 °C and 31 °C
acclimated fish. Cross-hybridisation
of regulatory
proteins (tropomyosin-troponins complex) from coldacclimated fish to desensitised actomyosin from
warm-acclimated fish alters the A TPase towards that
of cold-acclimated natural actomyosin and vice versa.
The results suggest that the regulatory proteins can
influence the kinetics of the A TPase and, furthermore,
that they are involved in the acclimation of the actomyosin to different cell temperatures.
Introduction
Following a period of acclimation goldfish show a
partial compensation in metabolic rate over a wide
range of environmental temperatures (Hazel and
Prosser. 1974; Smit et al., 1974). Temperature com-
pensation in fish is known to involve adaptations
in neural function (Bass, 1971 ; Lagerspetz, 1973)
membrane fluidity (Hazel, 1973; Cossins et al., 1977)
contractile proteins (Johnston and Walesby, 1977)
and energy metabolism (Hazel and Prosser, 1974; Hochachka and Somero, 1973; Somero and Low, 1977).
Recent studies have shown that cold acclimation is
associated with an increase, compared to fast glycolytic fibres, in the relative proportion of slow and
fast twitch oxidative fibres in the myotome (Johnston
and Lucking, 1978). In addition evidence has been
found for the synthesis 6f distinct kinetic forms of
Mg2 +Ca 2 + myofibi:illafA TPase at different environmental temperatures (Johnston et al., 1975; Johnston
and Lucking, 1978).
In vertebrate skeletal muscle myofibrillar A TPase
is inhibited in the absence of Ca 2 + by cooperative
effects of the calcium regulatory proteins located on
the actin filaments (Ebashi and Endo, 1968; Lehman
and Szent-Gyorgi, 1975). Arrhenius plots of rabbit
natural actomyosin A TPase show a break in slope
at around 15-18 °C (Bendall, 1969). In contrast, linear
Arrhenius plots are obtained for desensitized actomyosin from which the regulatory proteins have been
removed (Hartshorne et al., 1972; Fuchs et al., 1975).
Although this is not likely to be of physiological significance for a homeothermic animal such as the rabbit,
it does provide evidence that the calcium regulatory
proteins can modify the kinetic behaviour of actomyosin A TPase in addition to their traditional role
in excitation-contraction
(EC) coupling (Hartshorne
et al., 1972; Fuchs etal., 1975). In the present study
further evidence has been obtained that the thin filament Ca2+-regulatory proteins can influence the kinetics of actomyosin A TPase. The different forms
of goldfish actomyosin A TPase observed after a
period of temperature acclimation have been shown
to result at least in part from adaptations in the calcium regulatory proteins. To the author's knowledge
0340-7616/79/0129/0163/$01.00
164
I.A. Johnston: Go.Idfish Actomyosin
ATPase
this is the first reported example of a mechanism
by which a muscle fibre might intrinsically adapt its
speed of shortening other than by transforming a
fast to a slow tvoe of mvosin or vice versa.
of 90 mM KCI, 1.5 mM ATP, 2 mM MgCI2, 0.1 mM CaCI2,
pH 7.5. Temperature control was achieved bya magnetically stirred
thermostatically controlled water jacket ( :1:0.1 °C). In assays of
Mg2+ EGTA ATPase 1 mM EGTA replaced 0.1 mM CaCI2 in
the incubation medium.
In natural actomyosin preparations trace quantities of calcium
( ~ 10- 5M) are required to overcome the inhibition of the A TPase
Materials and Method"
by regulatory proteins of the tropomyosin-troponins
complex. Calcium sensitivity of actomyosin preparations was expressed by the
Goldfish (Carassius auratus. L.)approximately
50 9 in weight were
acclimated for 5 months in tanks of filtered, circulated fresh water
regulated to the following temperatures, 2 °C :t 0.5 °C (short photoperiod 8 h light: 16h dark) and 31 °C:t0.5 °C (long photoperiod
18 h lij(ht: 6h dark).
Preparation ~r Natural Actomyosin
Fish were stunned by a blow to the head and killed by decapitation.
White dorsal epaxial muscle was immediately dissected from the
trunk. Care was taken to avoid sampling superficial fast (pink)
and slow (red) oxidative fibres as these have been shown to have
different A TPase activities to the white fibres (Johnston et al.,
1977). The muscle was minced with scissors and homogenised at
0 °C with an Ultra Turrax blender for 2 x 40 sin 0.1 M KCI,
10 mM Tris-HCI, pH 7.0. The homogenate was centrifuged at
10,000 9 for 10 min and myofibrils prepared from the residue as
described previously (Johnston and Tota, 1974). Natural actomyosin was extracted from well washed myofibrils by 20 min gentle
stirring in 0.7 M KCI, 15 mM Tris-HCI, pH 7.0, 1-5 mM ATP
at 0 °C (Lehman and Szent-Gyorgyi, 1975). The insoluble residue
was removed by 15 min centrifugation at 20,000 g. The supernatant
was diluted to an ionic strength of 0.05 to precipitate the actomyosin. The precipitate was collected by centrifugation
at 20,000 9
for 10 minand resuspended and washed twice in 0.1 M KCI, 5 mM
Tris-HCI, pH 7.0. Experiments on the ATPase activity of natural
ac1:omyosins were performed on the same day as preparation.
Preparation of Desensitized Actomyosin
and ReKulatory Proteins
Desensitized actomyosin was prepared from natural actomyosin
as described by Schaub et al. (1967), and Dabrowska
and
Szpacenko (1977). Natural actomyosins were dialysed for 24-48 h
against several changes of 2 mM NaHCO3 at 0-4 °C. The actomyosin was collected by 10 min centrifugation at 20,000 9 and washed
a further 6-8 times with 20 volumes of distilled water. The supernatant was used to prepare crude calcium regulatory proteins complex
as described by Regenstein and Szent-Gyorgyi, (1975). The desensitized actomyosin was finally resuspended in 0.1 M KCI, 10 mM
Tris-HCI, pH 7.0. pesensitized actomyosin preparations were occasionally stored at -25 °C in a 50: 50 (v/v) solution of glycerol
and 0.1 MKCI, 10 mM Tris-HCI, pH 7.0. These preparations were
not calcium sensitive and examination of natural and desensitized
actomyosins on 10% SDS polyacrylamide gels (Weber and Osborn,
1969) showed that the washing procedure removed all bands corresponding to troponins and most of the tropomyosin (Fig. I). Preparations of crude regulatory proteins contained no myosin or material of chain mass in excess of 80,000 Dalton's but werecontaminated with actin
Determination.,
of A TPa",
Artj"jti",
Measurements of A TPase activities were made in a pH stat (Radiometer, Copenhagen) by monitoring H+ release in a medium
following
relationship:
Percentage calcium sensitivity ( %)
=
[(1-
Mg2+EGTA ATPaSe
Mg2+Ca2+ ATPase
) 100) .
Measurements of Mg2+Ca2+ ATPase activities were made at a
series of temperatures between 0 °C and 32 °C. Slopes and 5%
and 1% confidence limits of the linear regression of the corresponding Arrhenius plots were calculated by computer program. In cold
acclimated fish Arrhenius plots of natural Mg2 +Ca2 + actomyosin
A TPase show a break in slope at around 15-18 °C. Activation
energies (Ea = A H* + R7) were therefore calculated for the temperature ranges 0-16 °C and 16-32 °C. Protein concentrations were
determined using a standardised biuret method (Gornall et al.,
1949)
Results and Discussion
Conventional techniques used to isolate mammalian
myosins yield preparations of low enzymic activity
when applied to cold:.adapted fish due to the formation of aggregated and denatured products (Connell,
1961; 1969). However, actomyosins prepared from
the same muscles have high specific activities and
are considerably more stable. Therefore in the present
comparative study natura}. and desensitised actomyosin preparations were .used in preference to synthetic
actomyosins reconstituted from purified proteins.
Natural actomyosin contains all the proteins of the
calcium regulatory system in addition to actin and
myosin. Examination
of desensitised actomyosin
preparations on 10% SDS polyacrylamide gels revealed that the washing procedure employed to desensitise the A TPase to calcium removed all protein
bands corresponding to the troponins complex and
most of the tropomyosin (Fig. I).
Temperature acclimation in goldfish results not
only in an altered distribution of muscle fibre types
but also in the synthesis of different kinetic forms
of actomyosin ATPase (Johnston et al., 1975; Johnston and Lucking, 1978). In the present study, when
assayed at 31°C the Mg2+Ca2+ ATPase of natural
actomyosin was 4 times higher in 31 °C than 2 °C
acclimated fish p < 0.01 (Table 1). In contrast,
Mg2 +Ca 2 + A TPase activities of 2 °C and 31 °C acclimated fish were similar when assayed at 2 °C (Table 1). A somewhat different result was obtained by
Johnston et al. (1975) in which cold acclimated fish
had higher activities at lower temperatures than their
A. Johnston;
Goldfish Actomyosin
ATPase
165
2
65,000
Actin
45,000
TM
Q)
(/)
~
Coo
f<i
TNI
1
~
TNC
01
O
-.J
0
AB
Fig. 1. 10% SDS polyacrylamide gels of A natural and B desensitised goldfish actomyosin. Bands identified by running purified
goldfish muscle proteins included actin, tropomyosin (TM), troponin I (TNn and troDonin C (TNC)
Table I. Specific activities of Mg2 + Ca :
natural actomyosin
3.3
Assay
temperature
temperature
(OC)
(OC)
2
31
31
31
2
31
No. of fish
5
5
5
,
A TPase
activities
J.Lmoles
H+/mg/min.
3.6
3.7
1/T(OK)X103
AT.
0.085 : 0.013
0.50
: 0.08
natural actomyosin A TPase (Fig. 2). Arrhenius plots
of natural actomyosin A TPase from cold acclimated
gold fish show a break in slope at 15-18 °C (Fig. 2).
In contrast, linear Arrhenius plots are obtained for
warm acclimated actomyosin over the temperature
range 0-32 °C (Fig. 2). Cold acclimation is associated
with a reduction in activation enthalpy(AH*)
of the
ATPase (Table 2; p < 0.001). This is presumably advantageous for the cold adapted enzyme in reducing
0.095 : 0,.019
2.10 - 0.20
warm acclimated counterparts. It is not clear whether
this is due to different conditions of acclimation or
stock differences in the goldfish used in these experiments. However, in both studies acclimation resulted
in a modification of the temperature dependence of
Table 2. Energies of activation
and 31°C for 5 months
3.5
Fig. 2. Arrhenius plots ofMg2+Ca2+ ATPase activity (moles ATPI
mole myosin. sec) of natural actomyosin prepared from the white
skeletal muscle of goldfish acclimated to 2°C (open circles) and
31 oC (solid circles). Triangles represent desensitised actomyosins
to which calcium sensitivity has been restored by addition of an
excess of crude calcium regulatory protein complex. See text for
conditions of ATPase activities
Pase activity of !(oldfish white muscle
Acclimation
3.4
(Ea = JH* + R1) of natural
Preparation
No. of
fish
and desensitized actomyosin
Data
points
Temperature
Activation
range
cal/mole
Hot acclimated natural actomyosin
75
0-32
Cold acclimated natural actomyosin
31
26
0-16
16-32
47
Hot acclimated desensitized actomyosin
4
Cold acclimated desensitized actomyosin
~
A TPase activities
rC)
of fish acclimated
energy
Significance
of
600
to 2°(
regression
21,
000 :t
12,
350 :t 1350
P<0.01
10,
000 :t 1370
P<0.01
0-32
21,
500 :t 1100
P < 0.001
200 :t 1400
P<
P < 0.001
49
0-32
19,
Hot acclimated desensitized actomyosin
+ "hot" regulatory proteins
21
0-32
16, 350:!: 1240
Hot acclimated desensitized actomyosin
+ .'cold " regulatory proteins
16
0-16
9, 700 :t 1400
p < 0.05
16
16-32
4,900:t
1370
p <
0.05
26
0-32
13,700:t
870
p<
0.001
12
0-16
0.05
16-32
10,600:!: 1100
6.100+ 1050
p<
12
p<
0.05
Cold acclimated desensitized actomyosin
+ ., hot " regulatory proteins
2
Cold acclimated desensitized actomyosin
+ " cold " regulatory proteins
2
0.001
P < 0.001
.A. Johnston:
16(;
Il.
ATPa~
reconstituted natural actomyosin were lower than for
the native A TPase (Table 2). Cross-hybridisation of
regulatory proteins from warm acclimated fish with
cold acclimated desensitised actomyosins or vice versa
resulted in a reciprocal transformation of the temperature dependencies of the parent actomyosins (Fig. 4,
Table 2; p < 0.05). The results suggest that the regulatory proteins can influence the kinetics of the A TPase
and furthermore are involved in the acclimation of
the actomyosin to different cell temperatures.
Cross-bridge activation in vertebrate skeletal muscle is controlled at least in part by Ca 2 +-regulatory
2
Q)
{1)
(\3
Goldfish Actomyosin
1
1~
o
r5i
o
~
0
3.3
3.4
1/T
3.5
3.6
(OK)
x 103
3.7
Fig. 3. Arrhenius plots of desensitised actomyosin A TPase activities
(moles ATPsplit/mole
myosin.sec) of 2 °C (open symbols) and
31°C (solid symbols) acclimated goldfish
Q)
(/)
[0
0..
f«
At!
~
.~
A-.
Jt.
~ ,
..~
0
01
O
...
YI1
3.3
"
~"
" ~"
."
a. #. ~ ~
..~
,
I
I
I
,
3'4
3.5
3'6
3'7
1/T(OK)x103
Fig. 4. Arrhenius plots of hybridised actomyosins. Open triangles
represent A TPase activities (moles A TP split/mole myosin' sec) of
cold acclimated desensitised actomyosin
to which regulatory
protein complex from warm acclimated goldfish has been added
and closed triangles represent warm acclimated desensitised actomyosin plus cold acclimated regulatory proteins
the temperature sensitivity of the activation process
(cf. Low et al., 1973).
In contrast, desensitised actomyosins from which
the calcium regulatory proteins have been removed
show a linear temperature dependence over the range
0-32 °C and have similar properties regardless of acclimation temperature (Table 2, Fig. 3). Calcium sensitivity (60-70%) could be restored to desensitised
acto myosins by addition of crude regulatory protein
preparations (10: 1 w/w). Activation energies for the
system consisting of tropomyosins and troponins, located on the actin filaments. In the absence of calcium
« 10-7M) the tropomyosin-troponins
complex inhibits the activation of myosin A TPase by actin
(Ebashi and Endo, 1968; Weber and Herz, 1963).
Other effects of tropomyosin and troponin on the
interaction between actin and myosin have recently
been reviewed (Fuchs, 1974). The discontinuities in
Arrhenius
plots of rabbit
natural
actomyosin
Mg2+Ca2+ ATPase at 15-20°C have been shown to
result from the binding of Ca 2 + to its receptor site
on troponin C (Hartshorne et al., 1972). Arrhenius
plots of synthetic actomyosin which do not contain
the Ca2+-regulatory proteins are linear over the same
temperature range (Hartshorne et al., 1972; Fuchs
et al., 1975). The actin-activated A TPase of Limulus
skeletal myosin is increased several fold by the addition of tropomyosin (Lehman and Szent-Gyorgyi,
1972). A similar though less marked effect has also
been observed for rabbit myosin (Eisenberg and
Kielley, 1970; Shigekawa and Tonomura, 1972). In
addition, a recent study of tension generation by actomyosin threads has provided evidence that the thin
filament Ca 2 +-regulatory proteins can influence the
shape of the force velocity curve. Addition of tropomyosin-troponins to threads of synthetic rabbit actomyosin increased maximum isometric tension, by
40% and contraction velocity by 80% (Crooks and
Cooke, 1977).
The present study strongly suggests that the Ca2 +regulatory proteins are involved in the acclimation
of goldfish actomyosin to different temperatures.
However, adaptations in myosin are not excluded in
the present study. Interestingly, it has been shown
that the L2 light chain of myosin changes the ability
of myosin to interact with the thin filament in the
presence of calcium and decreases the Ca2 + concentration required to activate the thin filament (Pemrick, 1977). Furthermore, recent evidence has been
obtained for the presence of a myosin-linked Ca2 +regulatory system in vertebrate skeletal muscle (Lehman, 1978). However, adaptations in the kinetics of
goldfish actomyosin A TPase in the present study
I.A.
Johnston:
would
Goldfish
appear
Actomyosin
to require
A TPase
modification
167
of the Ca 2 + -
regulatory proteins irrespective of, or in addition
to,
any changes in myosin. This provides further evidence
that the thin filament
regulatory
proteins
are not
merely an " on-off"
switch for controlling
contraction
but can also influence the kinetics of myosin crossbridges following
activation
by calcium.
Further
studies are in progress to characterise
tropomyosin
and troponins isolated from fish acclimated to different temperatures.
'
This work was supported by grants from The Royal Society Scientific Investi~ations Fund and the ScienceResearchCouncil.
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