A. Johnston: Temperature Adaptation of Fish Sarcoplasmic Reticulum. Table I. Habitat ..... tween AH* and cell adaptation temperature have been demonstrated ...
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). *(!) o "' 0: UJ
0: 2 « ~ ti
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
