Molecular Mechanisms of Temperature Adaptation in Fish ... - synergy

Journal .T comn.

Phvsiol.

119.195-206

(1977)

of

Comparative

Physiology. (C) by Springer-

B V erlag

1977

Molecular Mechanisms of Temperature Adaptation in Fish Myofibrillar Adenosine Triphosphatases Ian

A.

Johnstonl

and

N.J.

Walesby2

1 Department of Physiology, University of St. Andrews, St. Andrews, Fife, Scotland 2 Life Sciences Division. British Antarctic Survey, Madingley Road, Cambridge, England Received January 5. 1977

Summary. Studies have been carried out on the Mg2+Ca2+-myofibrillar A TPase from the muscles of fish adapted to different environmental temperatures. The thermal stability of the A TPase is strongly correlated with mean habitat temperature. Activities of Antarctic fish A TPases are significantly higher at low temperatures than those of temperate and tropical water species. The effects of ionic strength on A TPase activity have also been studied. The Gibbs free energy of activation (JG*) was found to increase and enzyme activity decrease with increasing ionic strength within the physiological temperature range of each species. Significantly lower values of JG*, of around 1 Kcal/mole, are obtained for the A TPase of cold-adapted compared to tropical fish. Enthalpic and en tropic activation energies were also reduced in the cold adapted A TPases. It is postulated that the reduction of the enthalpic activation term in the cold adapted enzyme confers the advantage of reducing the temperature sensitivity of the rate limiting step thus partly compensating for the low heat content of the ce.1lular environment. Possible molecular mechanisms of temperature compensation in fish myofihrillar A TPases are discussed.

Introduction Poikilothermic vertebrates successfully exploit thermal environments in the range -2 °C to 40 °C. Many species show adaptive changes which allow them to exhibit similar rates of physiological activity in spite of widely different body temperatures. Evolutionary adaptation to relatively narrow temperature ranges has resulted in the selection of homologous enzymes each with characteristic physical and catalytic properties (Hazel and Prosser, 1974). Interpretation of comparative studies on the thermodynamic and kinetic properties of poikilothermic enzymes is often made difficult by the absence of reliable kinetic models. In the case of muscle contraction, a kinetic scheme for the hydrolysis of A TP by myosin subfragment 1 in dilute solution has been elucidated which involves at least

.

.A. Johnston

196

and N.J.

Walesby

7 intermediate steps (Trentham et al., 1976). Correspondingly detailed kinetic schemes relating to crossbridge cycling mechanisms and actin-myosin interactions in the intact myofibrillar matrix are not currently available for any system (Hill and Eisenberg, 1976). The uncertainties and complexities of kinetic modelling have confmed most comparative studies to determinations of catalytic rate constants (KcaJ of activation (Bendall, 1969; Low et al., 1973; Johnston and Goldspink, 1975). Although direct mechanistic interpretations of such studies are difficult, they have nevertheless proved useful in establishing a framework for investigating some of the underlying strategies of temperature adaptation in poikilotherms (Hochachka and Somero, 1973; Precht et al., 1973; Hazel and Prosser, 1974). In the case of fast twitch muscles of fish, the A TPase activity of myofibrils from cold adapted species is considerably higher at low temperatures than for tropical species (Johnston et al., 1975a). Differences in rate compensation between cold and warm adapted A TPases are reflected in changes in thermodynamic activation parameters (Johnston and Goldspink, 1975). In particular, the relative contributions of enthalpic and entropic activation terms to the free energy of activation varies according to environmental temperature. In a preliminary study it was concluded that reduction of the enthalpy term in the myofibrillar A TPase from cold-adapted muscles may confer an energetic advantage in reducing the temperature sensitivity of the activation process (Johnston and Goldspink, 1975). Small reductions in Gibbs free energy of activation for cold-adapted A TPases might also result in significant increases in reaction rate at low temperatures (cf. Low et al., 1973). In the present study activation parameters for the Mg2 +Ca 2+-activated A TPase have been compared for fish inhabiting different environmental temperatures. Since the A TPase activity is highly sensitive to ionic strength, the effect of this parameter on the rate and thermodynamic activation terms has also been investigated.

Materials and Methods Fi.h Two species of Antarctic fish were used in these investigations. Notothenia neglecta were caught by trammel net at Signy Island, South Orkney Islands. The single specimen of haemoglobinless "ice-fish" (Chaenichthyiidae), Champsocephalus gunnari, was obtained by otter trawl from 250 m depth off South Georgia, and was the first specimen of any ice-fish to be kept alive outside Antarctica. Fish were transported to the U.K. in tanks of filtered recirculated seawater maintained at loC. Indian Ocean species were obtained from local fish dealers and kept in aquaria at their habitat temperature (26 :t I °C) for several weeks before use. Specimens of the North Sea fish CoItus bubalis L. were obtained from local fishermen at Pittenweem, East Fife, during November and maintained

in tanks of recirculated

seawater at 4 °C.

Preparation of Myo{ibrils Fish were stunned by a blow to the head and killed by decapitation. Several grams of white epaxial muscle were immediately dissected from the trunk. The muscle was minced with scissors and homo17"n;""t1 "t ()Or with" P...lvtr...n hlpnt!pr f...r .'vM\. ;n 01 M11"rl 10~~A T..;oU£"'1

-

Temperature

Adaptation

in Fish Myofibrillar

A TPases

107

buffer at pH 7.0. The extent of homogenisation was monitored by microscopical examination. The homogenate was centrifuged at 10,000 x 9 for 10 min and the myofibrils prepared from the residue by the method of Perry and Qrey (1956). In preliminary experiments following preparation myofibrils were treated with a 1% solution of Triton X-I00 as described by Solaro et al. (1971). Treatment with Triton solubilises the sarcoplasmic reticulum and reduces possible contamination with membranous A TPases without affecting the myofibrillar A TPase activity (Solaro et al., 1971). The detergent was removed by washing 5 times in 50 vols. of 0.1 M KCI, 10 mM Tris-HCI, pH 7.0. The contribution of sarcoplasmic reticulum A TPases to the total measured activity in the original preparation was found to be negligible. Myofibrils were also tested for contamination by mitochondrial ATPases as previously described (Johnston and Tota, 1974). Preparations were essentially free of non-myofibrillar A TPases under the assay conditions employed. Myofibrils were finally suspended in the preparation medium at a concentration of approximately 10 mg/ml. Protein concentration was determined by a standardised biuret method (Gornall et al., 1949).

Enzyme Assay The standard assay for Mg2+Ca2+-activated myofibrillar ATPase was performed in a volume of I ml of 50 mM Tris-HCI pH 7.5,5 mM disodium ATP, 5 mM MgCI2, 0.1 mM CaCI2 at a myofibril concentration of 0.4-0.5 mg/ml and ionic strength of 0.10 (adjusted with KCI). Although the Mg2 + -activated A TPase activity is being studied trace quantities of calcium are required to overcome inhibition by the calcium regulatory proteins of the tropomyosin-troponins complex. Following Fuchs et al. (1975) we have used the designation Mg2+Ca2+-stimulated myofibrillar A TPase since calcium ions in low concentration have been found to reduce the activation enthalpy at temperatures greater than 15-20°C. The measured A TPase activity therefore closely parallels the in vivo physiological myofibrillar A TPase. The reaction was started by addition of A TP to preincubated myofibrils and terminated with I ml of 10% (w/v) trichloracetic acid. Precipitated protein was removed by centrifugation and inorganic phosphate measured in an aliquot of the supernatant by the method of Rockstein and Herron (1951). Appropriate enzyme and reagent blanks were included in all experiments. Preparations were tested for calcium sensitivity in assays in which 4 mM EGTA replaced added calcium (0.1 mM). Under these conditions the activity was usuallv less than 10% of that in the Dresence of trace amounts of calcium.

Ionic StrenJ!th Exoeriments Assay conditions for ionic strength experiments were as follows: 25 mM Tris-HCI pH 7.5, 5 mM MgCI2, 5 mM ATP, 0.1 mM CaCI2, 0.5 mg/ml myofibrils with the remainder of the ionic strength (11) being contributed by sodium-p-glycerophosphate, this being used in preference to KCI or NaCI since chloride ions have been shown to have an inhibitory effect on the A TPase activity at very low ionic strengths (11= 0.1) (Bendall, 1964).

Therma/ Inactivation

Experiments

Thermal inactivation of the Mg2+Ca2+-activated ATPase was carried out in a w1tter-jacketed reaction vessel fitted with a magnetic stirrer in a medium of 0.05 M KCI, 40 mM Tris-HCI at pH 7.5. Myofibrils (0.8-1.0 mg/ml) were added to an 18-fold excess of medium previously incubated to 37°C. An initial sample was taken within 3 sec and subsequently at appropriate intervals and pipet ted into tubes cooled in melting ice to prevent further inactivation. Myofibrils partially inactivated by exposure to high temperatures were assayed for ATPase activity at 18°C.

Ca[culation or Thermodvnamic Parameters Mg2+Ca2+-activated ATPase activity was measured in duplicate at a series of temperatures. Arrhe. nius plots of fish Mg2+-myofibrillar ATPase often show a transition break at 15-18°C (Bendall,

I.A. Johnston and N

IQR

Wale.hv

1969; Johnston et al., 1973). In addition, the Mg2+Ca2+-ATPase of cold-adapted myofibrils shows an initial activation and subsequent rapid thermal denaturation at temperatures in excess of 28-29 oC (Johnston et al., 1973, 1975a). For this reason measurements of ATPase activity were made for the temperature ranges 0-15°C and 18-28°C. Apparent activation energies (AH*+RT) over these temperature ranges were calculated from the slopes of the corresponding Arrhenius plots. Thermodynamic activation parameters were calculated by the following relationships as described by Lehrer and Barker (1970): AG* = AH* -TAS* AH*=E

. -RT

AS* =4.576 r1ogto k-10.753-logtnT+-",

1:.. .: 4.5761..

The rate constant k (s- I) is proportional to v max and expressed as moles A TP split/mole enzyme active site/second. The proportion of myosin in the myofibril was assumed to be 54% (Bendall, 1969) with a molecular mass of 240,000 daltons per enzyme site (Lowey et al., 1969; Godfrey and Harrington, 1970). Statistical analyses were carried out using analvses of variance for eQual sample numbers.

Results and Discussion Thermal Stabilitv A measure of the thermal stability of the active site was obtained by assessing the loss of A TPase activity at 37 oC. In some cases the inactivation of the Mg2+Ca2+-activated ATPase followed first-order reaction kinetics (Fig. la, b, c). However, in most warm-adapted enzymes there was an initial increase in activity following preincubation at 37°C (Fig. le). It would appear that the initial stages of thermal denaturation result in a conformation which is more active than the native enzyme. Similar transient increases in activity resulting from unfolding of the native enzyme have been observed in fish myosins following denaturation by urea and in the thermal denaturation of sarcoplasmic reticulum Ca 2 +-A TPase activities (Syrovy et al., 1970; Carvalho and Santos, 1976). The half-life of inactivation of the Mg2+Ca2+-activated myofibrillar ATPase was positively correlated with adaptation temperature. Three Antarctic species investigated all showed a half-life of inactivation of less than 1.5 min. The order of stability Notothenia rossii > Champsocepha/us gunnari > Notothenia neg/ecta was correlated with degrees longitude south, the most labile A TPase coming from the most southerly species. This compares with .a half-life of inactivation of around 10 min for North Sea species (environmental temperature 5-12°C) and 60-100 min for Indian Ocean species (environmental temperature 20-25 OC). The variation in "stability" of the Mg2 +Ca 2 +-A TPase according to environmental temperature is illustrated in Figure 2. The most stable enzyme so far investigated is that from Ti/apia grahami a species living at 36-40 oC in a hot-springs soda-Iake in central Kenya (tl/2 at 37 oC = 520 min) (Johnston et al., 1973). There have been numerous reports of protein or cell thermostability being correlated with habitat temperature in poikilotherms (Ushakov, 1964; HH7el and Prosser. 1974). It has been su~gested that in some cases heat dama~e

.

Temperature

Adaptation

in Fish Myofibrillar

199

A TPases

"

~

~= ~ .t. u =0

3,4

3,5

3,6

3,7

I/T"K x 103 Fig. 3. Arrhenius p!ot of )oglo Mg2+Ca2+-activated ATPase activity (moles ATP split.mole , myosin active site- I. s- ') against liT (OK x !03) for white muscle myofibrils of the icefish, Champ- . socephalus gunnari (ET -! oC to +2°C). text. Significance of regression P ~

= ~ ~ u ~ 0

3,3

3,4

3.5

l/r'K

3,6

3.1

x 103

Fig, 4, Arrhenius plot of log10 Mg2+-Ca2+-activated ATPase activity (moles ATP split.mole myosin active site- I.S- 1) against I/T (OK x 103) for white muscle myofibrils of an Indo-Pacific species Pomatocentrus uniocellatus (ET + 18 °C to + 26 °C). Mean of two preparations. Assay conditions given in text. Regressions over the ranges o°C to + 15 °C and + 18 °C to +28 °C sil!nificant at the P=O.OI level

.

"'"--

Temperature Adaptation

in Fish Myofibrillar

A TPases

203

tropical species (P < 0.001; Tables 1 and 2). This will have the effect of increasing the proportion of molecules with sufficient energy to form the activated complex at any given temperature. Arrhenius plots for the Antarctic species Champsocephalus gunnari were linear in the range 0-28°C (P