Download Evolutionary Temperature Adaptation of Fish Sarcoplasmic Reticulum

Journal
J. Comp
Physiol
~~157-lh4(IQR()\
of
Comparative
Physiology.
B
j
1980
by Sprin.L!er-Verla.L!
Evolutionary Temperature Adaptation of Fish Sarcoplasmic Reticulum
Harry
J. McArdle
Department
and
Ian
A.
Johnston
of Physiology. University of St. Andrews, St. Andrews, Fife. Great Britain
Accepted October 10.1979
Summary. Sarcoplasmic
reticulum
has been isolated
from the white muscle of 15 species of teleost fish
adapted to diverse thermal environments.
Evidence
has been obtained that the Ca 2 + -dependent A TPase
of fish
tionary
atures.
species
sarcoplasmic
reticulum has undergone evolumodification
for function at different temperCompared
with tropical
fish, cold adapted
have higher rates of Ca2+ transport and Ca2+ -
A TPase activities at low temperatures.
Most species
have linear Arrhenius
plots over the temperature
range 0-30 °C. Activation
enthalpies
(.1H*) of the
A TP~se ranged from 53-190 kJ mol- 1 and were positively correlated with environment
temperature.
Activation entropy (.1S*) varied from negative values in
cold
fi!;h.
adapted
species to positive
In contrast
values
in tropical
to the Ca 2 + -A TPase, the " basal"
AT -
Pase of fish sarcoplasmic
reticulum
showed no relationship between either A TPase activity or thermodynamic activation
parameters and environmental
temperature.
Only the Ca 2 + -dependent A TPase is coupled to
Cil2 + transport.
The percentage of ..total " A TPase
activity
which
is Ca 2 + activated
is higher
at low tem-
peratures in cold than in warm adapted species. For
example, ratios of Ca2+ -dependent/total
A TPase at
2 °C varied from 80-98%
in Arctic,
Antarctic
and
North Sea species to only 2-50% in various tropical
fish. Above 20 °C, similar ratios in the range 80-98%
were obtained for all species. The nature of the " basal" A TPase and mechanisms of temperature
adaptation of fish sarcoplasmic reticulum are discussed.
Ahhreriali{)n.~:
glycol-bis
ET.
environmental
(p-aminolethyl
ether)-N.
N-2-hydroxylpiperazine-N'.2-ethanesulfonic
mic reticulum
temperature;
N'-tetraacetic
acid;
EGTA,
ethylene
acid;
HEPES.
SR.
sarcoplas-
Introduction
A number of studies have shown that cold adaptation
in fish is associated with an increased incorporation
of unsaturated fatty acids into membrane phospholipids (Johnson and Roots, 1964; Knipprath and
Mead, 1968; Hazel, 1973; Cossins, 1977; Cossins
et at, 1977). This is thought to provide a mechanism
for controlling membrane fluidity and hence conserving membrane function at different temperatures
(Hochachka and Somero, 1973; Sinesky, 1974; Hazel
and Prosser, 1974; Chapman, 1975). For example,
Cossins and Prosser (1978) have shown a correlation
between phospholipid unsaturation, membrane viscosity, and adaptation temperature for brain synaptosome preparations from a variety of species of fish
and small mammals. Interestingly, no such relationship was evident for membrane fluidity of sarcoplasmic reticulum (SR) membrane (Cossins, 1977). Furthermore, there was no change in the phospholipid
unsaturation index of goldfish SR following acclimation to either 5, 15 or 25 °C, and only a poor correlation between fatty acid unsaturation and cell temperature for rat, desert pupfish and Arctic sculpin (Cossins. 1977; Cossins et al., 1978). This is a somewhat
surprising result in view of the known involvement
of phospholipids in the mechanism of Ca 2+ transport
(Meissner and Fleischer, 1974; Knowles et at, 1976).
The extent to which Ca2+ transport by SR has
undergone evolutionary modification for function at
different temperatures is unknown. The current study
investigates the effects of temperature on calcium
transport and the Ca 2+ -dependent A TPase activity
of native SR vesicles isolated from fish adapted to
a wide range of thermal environments.
Preliminaryaccountsof this work havebeenpresented
to the BiochemicalSocietv(McArdle and Johnston.1979a.b).
0340-761618010135101571$01.60
,~
H.J. McArd!e
158
Table
Temperature
Adaptation
of Fish Sarcoplasmic Reticulum
I
No. of
fi~h u~ed
Species
Nvlvlh£'nia
Hippv~ltlssvide,~
Gadu,~ ac~/iflnus
Plcurll11el'les
plalc,~svide
plalcs,~a
M)'v,\vcephalu~
~I'vrpiu,~
Da,~c)'llu,~ me/anarus
Echidna
nchu/v~a
Sah'e/inu,~
a/pinll,~
,~a/ar
O,~lronvlus
oce//alu,~
Osphrvnemus
~lIram)'
C0/0,~,~vma ~pp,
Ti/apia
mariac
Ti/aoia
mossambicca
Code
No.
Habitat
6
marine
7
marine
8
marine
9
freshwater
10
freshwater
11
freshwater
12
freshwater
13
freshwater
14
freshwater
fr",hw,,!er
I,
of 40 mM
or A TPase AClivitie,
A TPase
Tris-HCI,
of SR protein.
activity
was
25 mM
and Ca2+-EGTA
free calcium
concentration
Ca 2 + -independent
\
in the same medium.
measured
KCI,
5 mM
buffer
that
I mM
Ringvass"y. Norway
Almondbank.
Perthshire
South America
South America
South America
African lakes
African 'lakes
(OC)
6-4
2-10
2-10
2-10
2-18
2-18
22-28
22-28
2-8
2-12
15-28
15-28
15-28
24-30
24-30
The reaction was initiated by the addition of A TP (final
concentration 2 mM) to the pre-incubated incubation medium, and
terminated by adding an equal volume of 10% trichloroacetic acid.
Denatured protein was precipitated by centrifugation.
Inorganic
phosphate in the clear supernatant was determined by the method
of Rockstein and Herron (1951). Incubation temperatures were
controlled to within + 0.1 °C in a water bath.
at
10 QC in
MgCI2.
0.2-0.4
a medium
mg mI-l
of pH 7.2 to give
The rate limiting step of the calcium transport is thought to be
the hydrolysis of an ADP-insensitive
acid-stable phosphorylated
enzyme intermediate (Shigekawa and Dough~rty, 1978; Shigekawa
and Akowitz, 1979). Levels of the intermediate were measured
as described by inesi et al. (1970, 1976) in the total ATPase assay
medium. The reaction was started by the addition of (y32p)-A TP
(final concentration 2 mM, 0.4 ~Ci mi-l), incubated for 60 s, and
quenched by the addition of an equal volume of 10% trichloroacetic acid. The precipitated material was washed twice in
50 vols. of acidified incubation medium by centrifugation and resuspension. The final sediment was dissolved in 10 ml of scintillant
(100.100.5 toluene:Triton
X-100:Scintol
2 (Koch-Lite
Labs.»,
"nrl "mlnt.erl in a Packard Tri-Carb scintillation counter.
Determination
of Rate Constants
nnd Th"rmndvnnn,ir
Arfi,Jnfinn
Pnrnm"f"r'
An apparent rate constant k (s- 1) was obtained by dividing the
Ca 2+ -dependent A TPase activity by th() corresponding steady state
concentrations of the Rhosphoenzyme intermediate.
Activation energiet(E.)
of the Ca2+-dependent ATPase were
calculated from Arrhenius
plots of log k against liT (OK).
Thermodynamic
activation parameters were obtained according
to transition state theory from the following equations (Hidalgo
et al.. 1976):
a final
of 50 ~M (Heilmann
et al.. 1977).
basal)
A TPase activity
was measured
except
Environmental
Measurement of the Phosphorylated En:).'me
Tnlprmpdiatp
Reliculum
All operations were performed between 00 and 4°C. Fish were
killed by stunning and decapitation. and the white epaxial muscle
was rapidly excised. taking care to remove all traces of red and
intermediate muscle. The chopped muscle was homogenised in
3 vols. (w/v) of 0.3 M sucrose. 10 mM imidazole. pH 7.3. using
an Ultra- Tu.rrax blade homogeniser. at 3/4 full speed. for 3 x 20 s.
Myofibrils and cellular debris were pelleted by 30 min centrifugation at 2.500 g. The supernatant was centrifuged at 15.000 9 for
30 min to precipitate the mitochondria. The microsomal pellet was
obtained from the supernatant by centrifuging at 95.000 9 for 1.5 h.
The microsomal pellet was resuspended in the homogenising medium (10 ml) and layered onto a sucrose gradient consisting of
5 ml 40% sucrose. 10 ml 35% sucrose and 10 ml 30% sucrose.
in 10 mM imidazole. pH 7.3.
The gradient was centrifuged at 95.000 9 for 2 hand fractions
sedimenting between the homogenisation medium and 35% sucrose
were combined and used in all experiments. Preliminary experiments showed no appreciable contamination with either mitochondrial or sarcolemmal A TPases in this fraction (McArdle and Johnston, 1979b).
"Total
Balsfj"rd. Norway
Balsfj"rd. Norway
Balslj"rd. Norway
Firth of Forth. Scotland
Firth of Forth. Scotland
Indian Ocean
Pacific Ocean
marine
The species used in these experiments. their geographical location.
habitat and environmental temperature range are listed in Table I.
Where necessary. fish were maintained in filtered. recirculated
water at their habitat temperature.
Measuremenl
British Antarctica
marine
marine
",_1-
of Sarcoplasmic
marine
marine
Materials and Methods
Preparalion
Geo~raphicallocation
temperature
1
2
3
,1
2
6
2
2
6
6
2
2
6
4
6
2
2
2
2
rvs,~ii
Gadu,~ mvrhua
Sa/mv
and [.A. Johnston:
EGTA
replaced
k=(k.T/h)e~..G.IRT
(1)
the Ca2+AH*
EGTA
buffer.
Ca2--dependent
ATPase activity
was obtained
o..k..n~.;rtn
.kA k,.0.1 f.n~
tkA tnt.1 11TP"... ",.t;v;t;...
=E
by
AG*=AH*-T,jS*
.
-RT
(2)
(3)
~~
u
McArdle
(.I
o
and I.A.
Adaptation
of Fish
Sarcoplasmic
Reticulum
159
10
"
~
~
o
O
3
~
:
Temperature
~
;
..
~
0-
Johnston:
O
..1
100
====!'==
'<
,
.
"~
(.I
0
}F'-==-=+==
,a..=-:_-:.-=-~-f~:;;
'3
~~
"
10
20
30
Environmental Temperature ( .C )
Fig. I. Ca2+.ATPase
temperature
number
tion.
range
(1-15)
Activity
species
activity
at O °C is shown
normally
experienced
by
refers to a different
species.
is in nmole
protein/min.
used is given
P;/mg
in Table
plotted
each
See Table
against
species.
the
Each
I for explana.
Numbers
of each
0..
~
c
(
I
I/T.K (x10',
Fig. 4 A-D. Examples of Arrhenius plots for four species of teleost ;
A Notothenia rossii; B Salvelinus alpinus; C O.~tronotu.~ocellatus;
D Tilapia mariae. See Table I for details of environmental temperature See text for details of assav methods and conditions
20
40
Time
Fig. 2. Ca2+-uptake
lated
from
T;lap;a
(nmole
Notothen;a
mossamb;cca
Ca2+ /mg
ross;;
( .).
where" and h are Boltzmann's and Planck's constants, respectively
Values were computed at 2 °C (275 °K). Best-fit lines were corn.
mins
protein)
(. ). Pleuronectes
See text
for
details
at 0°C
by SR iso-
plalessa
puted using linear regression analysis.
(A ). and
of assay conditions
and methods
Calcium Uptake
Calcium uptake into isolated vesicles was measured using a Millipore filtration technique (Tume and Hunington, 1974). Vesicles
were pre-incubated in standard total ATPase medium containing
10 mM oxalate and 0.02 ~Ci/ml 45Ca2. .The reaction was started
by the addition of A TP to a final concentration of 2 mM. Aliquots
were taken at various times (0.5-50 min) and filtered through
0.45 ~m Millipore filters. The filters were washed with 2 x 2 ml
of cold incubation medium. air-dried. dissolved in 10 mi of scintillant (see above) and counted. Appropriate controls were included
2.0
in all exDeriments.
1.0
Protein
5
Protein concentrations were estimated using the modification of
the Lowry method (Lowry et al.,. 1951) proposed by Maddy and
15
10
E.5timatj
Spooner ( 1970).
Time
Fig. 3. Ca2+
uptake
isolated
Nololhenia
Ti/apia
from
mossamhicca
and methods
mins
(~mole
( .).
rossii
Ca2+jmg
(.).
See text
protein)
P/euronecles
for
details
at
25°C
p/alessa
by
(A).
SR
and
.\'tati.~tical Anah'ses
of assay conditions
St"ti~tical
analyses were carried out using the Student's (-test.
H
160
McArdle
and I.A. Johnston.
Temperature
Adaptation
of Fish Sarcoplasmic Reticulum
Fig. 5. Activation enthalpy values
(kJ mole-') of the Ca2+-ATPase for 15
species of teleost fish plotted against
geographical location. Species have been
divided into marine and freshwater fish.
The triangles at the top of the graphs
represent the approximate temperature
range experienced by each species. The
explanation of the numbers is given in
Table I. See text for details of assay
conditions and methods
.
Arctic
Sc~land
S America
Africa
Results
__1~-
*(/)
~
The Effects of Temperature on Calcium
and Ca2 + -Dependent A TPase Actirities
Uptake
The Ca 2+ -dependent A TPase activity of SR from 15
species of fish has been determined at a series of
temperatures between 0 and 30° C. Figure 1 shows
the Ca 2+ -A TPase activity at 0 °C plotted against the
environmental temperature (ET) range experienced
by each species. In general, cold adapted fish have
higher activities than warm-adapted species. Similarly
at 0 °C, the SR from two cold-adapted species Notothenia rossii (ET 0-4 °C) and P/euronectes p/atessa
(ET 2-18 °C) accumulate calcium at six times the
rate achieved by Ti/apia mossambicca (ET 24-30 °C)
(Fig.2). At 25 °C similar rates of Ca2+-uptake are
>
Q
e
c
w
0.1
c
9
iU
~
u
~
3.5
~~
6
9
0
.~
'3
=~~-=
---
4
=~-:---:::
2
20
10
30
Environmental Temperature ( 'C )
Fig. 6. Activation entropy values(kJ mol-l K -I) oftheCa2+-A TPase
are shown plotted against environmental temperature. Species 7,
8 and 10 are not included as there was insufficient sample material
to determine the steady state level of the phosphorylated enzyme
intprmprli"te
Ac\'* values were determined as described in the text
obtained for all three species (Fig. 3).
*(!)
<J
Thermodvnamic
Activation
a) The Ca2+-Dependent
Parameter!
ATPase
With the exception of Tilapia mariae. Arrhenius plots
of the SR Ca 2+ -A TPase were linear over the temperature range at 0-30 °C (Fig. 4). Presence or absence
of a discontinuity in the Arrhenius plot was not, therefore, correlated with any particular environmental
temperature. Steady state levels of the phosphoenzyme intermediate are independent of assay temperature, in the range 0.8-3.6 nmole Pi/mg protein, and
show no significant correlation with adaptation tem-
>o
"'
0:
UJ
0:
2
«
~
ti
<
70
2
'-~=:
:.--
11
14
+ -12--1;-
--==---
~~~!~3
5_6
~
65
60
.
10
20
EnvIronmental
Fig.7.
Activation
energy
values
are shown
against
environmental
10 are not
included
as there
determine
mediate
steady
Values
state
30
Temperature
levels
( .C )
(kJ mol-l)
of the Ca2+-ATPase
temperature.
was insufficient
Species
sample
of the phosphorylated
were determined
as described
7, 8 and
material
enzyme
in the text
to
inter-
HI
McArdle
A. Johnston.
and
Temperature
Adaptation
of Fish
Sarcoplasmic
161
Reticulum
"0
E
-,
~
.X
~
MArIne
,100
~
'i
~
~
UJ
.3
Fig. 8. Activation enthalpies (kJ mol- 1) of
the basal A TPase are plotted against
environmental temperature for 15 species of
teleost fish. Values of L1H* were
determined as described in the text.
.,
..
..
2
~
U
~
.,
50
I
Antarctic
N
ArctIc
5@.
Arct,c
IndoPaclf,C
I
Scotland
.f""A
S AmeriCa
2
100
.~;~--*;;========3:
_'=.'=.===t:==
.
..
>
1:'
12
13
10
~
0>
.,
If
~
~
11 +
;;?
50
,.
;
10
-20
;'2"
--0>
.,
If
~
:~
0
---:;0-
Env,'onmenta'Tampe,atu'e 1°CI
Fig. 9. The ratio
(in %) at 0°C
of Cal + -dependent
is plQtted
ities were determined
against
A TPase/tota]
environmental
as described
A TPase
temperature.
activity
Activ-
in the text
10
perature. Activation enthalpies (LIH*) of the A TPase
ranged from 53-190 kJ/mole and were positively correlated with environment temperature (Fig. 5). Activation entropy (LIS'*') varied from negative values in
cold adapted species to positive values in the tropical
fish (Fig. 6).
Values for activation free energy (LIG*) were not
strongly correlated with environmental temperature
und were in the range 64-69 kJ/mole (Fig. 7).
20
AssayTemperature
(-C)
Fig. 10 A and B. The ratio of Ca 2 + -dependent/total
A TPase activity
(in
for
%)
of fish:
'iuides:
at different
assay
temperatures
two
species
A the ratio for a cold-adapted
fish, Hippog/os.soide.s
B the ratio for a warm-adapted
species. Ti/apia
p/ate.smariae
In contrast
lation
(LIH*)
to the Ca 2 + -dependent
A TPase, no corre-
was observed
between activation
of the basal ATPase and environment
ture (Fig. 8).
enthalpy
tempera-
is shown
The Effect
of Temperature
on the Ratio
or Ca2 + -Dependent
to Total A TPase Activities
Ratios of Ca 2+ -dependent/total
b) Basal A TPase
30
A TPase activity
at
0 °C were correlated with the environmental temperature range of each species (Fig. 9). Cold adapted
species had ratios in the range 75-98% at this temperature compared with only 2-45% for the tropical
species. Above 20 °C, all species had similar ratios
H
162
McArdle
and I.A.
in t~e range 80-98%. The effect of assay temperature
on the ratio of Ca 2 + -A TPase/total
A TPase of a coldwater and tropical
fish is shown
in Fig. 10.
Discussion
It appears that fish sarcoplasmic reticulum has undergone evolutionary modification for function at different temperatures. The higher rates of calcium transport and A TP hydrolysis at low temperatures in cold
adapted species parallels functional adaptations in
catalytic efficiencies observed for other enzymes of
energy metabolism in fish muscle (Johnston et al.,
1973; Low et al., 1973; Somero and Low, 1976; Johnston and Walesby, 1977, 1979). Activation enthalpy
(AH*) of the Ca 2+ -A TPase is positively correlated
with environmental temperature for the 15 species
of teleost fish investigated. Similar correlations between AH* and cell adaptation temperature have been
demonstrated for fish muscle pyruvate kinases (Low
and Somero, 1976) and Mg2 +-Ca 2+ -myofibrillar
ATPases (Johnston et al., 1977; Johnston and Walesby, 1977, 1979).
Adaptations in AH* are associated with energetically unfavourable but biologically advantageous adjustments in activation entropy (Low and Somero,
1976; Johnston et al., 1977). Activation entropy (AS*)
varies from negative values in cold adapted fish to
positive values in more warm adapted species (Fig. 6).
Mechanistic interpretation of these results is made
difficult by a lack of information
concerning the
detailed kinetics of the reaction. It has been suggested
that these adjustments may result from differences
in weak bond formation during the activation process
(Somero and Low, 1976). In the case of the Ca 2+ A TPase of SR, weak bond formation might include
protein-protein,
protein-phospholipid
and membrane-solute interactions. In membrane-bound enzymes, the physical state of associated lipids may play
an important role in stabilising protein structure. For
example, in rabbit SR, below 17 °C there is a decrease
in membrane fluidity as detected by ESR spectroscopy of spin labels attached to both the A TPase
protein and SR phospholipids (Eletr and Inesi, 1973;
Davis et al., 1976; Hidalgo et al., 1976). This is associated with an inhibition of the A TPase activity, and
an increase in AH* and AS* (Hidalgo et al., 1976).
Replacement of endogenous phospholipids with more
unsaturated analogues results in an increase in both
membrane fluidity and A TPase activity at low temperatures (Warren et al., 1974; Hidalgo et al., 1976).
It might be expected, therefore, that adaptations in
catalytic efficiencies of cold adapted SR A TPases
would result from modifications both in orotein struc-
Johnston:
Temperature
Adaptation
of Fish Sarcoplasmic
Reticulum
ture and of the lipid microenvironment of the enzyme.
In the SR, however, the evidence for this is somewhat
con tradictory .F or example, C ossins et al. ( 1977) ha ve
shown that the fatty acid composition of SR phospholipids becomes unsaturated in the .order rat, desert
pupfish (ET 28-34 °C), and artic sculpin (ET
0.5-2 °C), but that there were no corresponding
changes in membrane viscosity. In contrast, other
studies have shown lobster SR to have a higher AT Pase activity at low temperature, and be more fluid
than SR from rabbit muscle (Morse et al., 1975;
Madeira and Antunes-Madeira, 1976).
The extent to which the discrepancies in the above
results represent phylogenetic diversity or differences
in the binding sites of the various fluorescence and
spin probes used. is unknown. Further work is required to elucidate the relative importance of adaptations in the protein and lipid components.
Associated with crude microsomal preparations
from skeletal muscle is an A TPase activity which is
not dependent on the presence of Ca2+ ions. Although this activity is generally referred to as the
basal A TPase it seems likely that it results from more
than one enzyme species. Some workers have thought
that the basal A TPase simply represents contamination with other membrane components (eg. T -system
tubules) (Headon et al., 1977), while others (eg. Inesi
et al., 1976) have suggested that it is a form of the
pump protein uncoupled from calcium transport.
In support of the latter hypothesis, treatment of
rabbit SR with a non-ionic detergent, Triton X-lOO,
results in conversion of Ca2+-independent to Ca2+dependent A TPase. In addition, the ratio of Ca2 +dependent to Ca2+-independent ATPase is temperature sensitive, being approximately 0.5 at 4 °C and
9.0 at 40 °C. Thus these workers envisage that the
SR A TPase enzyme exists in two states, in equilibrium
(E1~E2). The lower ratio at 4 °C is thought to be
due to the increased order (decreased fluidity) of
phospholipids stabilising the A TPase active site. Increasing the temperature increases the proportion of
phospholipids in the membrane which adopt the state
required for full activation of the A TPase and its
transport function.
Our observations on fish. SR suggest that both
hypotheses have some validity. Table 2 shows the
distribution of A TPase activities associated with microsomal fractions separated on a discontinuous sucrose gradient. The fraction sedimenting between
11% and 30% sucrose has almost entirely Ca 2+-independent A TPase activity. Incubation of these vesicles
in the presence of 0. I % Triton X-IOO does not result
in the conversion of Ca2+-independent to Ca2+-dependent A TPase (Table 3). It seems likely, therefore,
that the A TPase associated with this fraction arises.
-.
H.J.
Table
McArdle
and
I.A.
2. The distribution
of microsomal
discontinuous
sucrose
HEPES
buffer
isolated
gradient.
described
replaced
Fraction
Temperature
of A TPase activity
fractions
under the conditions
that
Johnston:
from
(nmol
Ti/apia
of Fish Sarcoolasmic
Pi mg-t
min-
mvs.famhica
The assay was performed
in ..Materials
Tris
')
on
a
at 20 °C
and Methods
..except
buffer
Ca 2 + -
Total
A TPase
1% sucrose)
Adaptation
Ca2+-
independent
ATP"""
252:t 16
218:t34
1.1~+'(;
dependent
A TPase
36:!:
Triton
X-100
Ca2+
total
11-30
5
dependent
ATPase
163
This work was supported by a grant from the SRC to Ian A.
Johnston. H.J. McArdle gratefully acknowledges receipt of a
University Scholarship. We are grateful to the Camegie Trust for
the Universities of Scotland. the Russell Trust (H.J.M.) and the
Royal Society European Scientific Exchange programme (I.A.J.)
for travel funds to visit the University of Tromso, Norway.
We thank Professors 0. Vahl and Geoffrey Wallace for the
provision of facilities at the Fisheries Institute, University of
Troms0. Antarctic fish were kindly donated by the British Antarctic
Survev
II
593:!:
136
(i.l2+
R
Table 3. The effect of Triton X-IOO (0.1 %) on the ratio of Ca2+ dependent A TPase/total A TPase activity of microsomal fraction
isolated from Ti/apia mossambica on a discontinuous sucrose gradient. The assay was performed at 20 "C under the conditions
described in ..Materials and Methods ...except that HEPES buffer
replaced Tris buffer. n.s. = not si~nificant at P=0.05 level
Fraction
% sucrose
Reticulum
p
A TPa...
0.16j :0.05
11-30
0.19:! :0.13
30-35
0.63:! :0.06
30-35
0.97:! :0.02
<11.001
35-40
1~-4()
0.80:! :0.03
11Q4.. .(} (}3
<0.05
at least in part, from membranes other than the sarcoplasmic reticulum.
There is evidence that the Ca l + -independent AT Pase of the other fractions is of a different nature.
Firstly they show negligible contamination with either
mitochondrial
or sarcolemmal A TPases (McArdle
and Johnston, 1979b). Secondly, the Cal+-independent ATPase of these fractions is converted to Cal+dependence by treatment with Triton (Table 3). Thus
the basal activity of these fractions may correspond
to an interconvertible form of the Cal+ -A TPase not
coupled to transport. As might be predicted from
this hypothesis, the ratio of Cal +-dependent to total
A TPase activity is higher at low temperatures in cold
than in warm adapted species (Fig. 9).
If this interpretation is correct, then any analyses
of the thermodynamics of the A TPase must consider
not only the free energy of activation of enzyme catalysis, but also the free energy of activation of the
conversion El to El. This may account for the poor
correlation observed between the apparent free energy
of activation (AG*) and environmental temperature
(Fig. 7).
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