Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 19 to 23. Peroamon Press. Printed in Great Britain
LACTATE D E H Y D R O G E N A S E M4 OF AN ABYSSAL FISH" STRATEGIES FOR F U N C T I O N AT LOW TEMPERATURE A N D HIGH PRESSURE JOHN BALDWINt, K. B. STOREY2. AND P. W. HOCHACHKA2 t Genetics Department, Research School of Biological Sciences, The Australian National University, Canberra, Australia 2 Department of Zoology, University of British Columbia, Vancouver, B.C., V6T 1WS, Canada (Received 11 October 1974)
Abstraet--l. Lactate dehydrogenase M 4 purified from the abyssal fish Antimora rostrata displays lower AG:~ characteristics and a higher turnover number than the homologous enzyme of homeotherms. 2. Pressures of 8000psi have no effect upon Vm~x, and the volume change of activation (AV:~)between 14.7 and 8000psi is negligible. 3. The apparent Km of the enzyme for pyruvate is insensitive to pressure at low temperature. 4. These properties are clearly compatible with function at the low temperature and high pressure environment of the abyssal floor.
INTRODUCTION
Low TEMPERATUREand high pressure are two environmental parameters which exert a direct influence upon enzyme function in deep sea fish. The effect of temperature and pressure on enzyme catalysis can be conveniently divided into two classes: (1) rate effects, which determine how rapidly equilibrium is attained between substrates and products; (2) weak-bond structural effects, which influence those properties of the enzyme that depend upon non-covalent bonding. Examples of this second class are 3° and 4 ° structure and the binding of substrates and modulator molecules. The effect of temperature on enzyme reaction rates is described by the Arrhenius equation: k
=
Ae -E/RT
where k is a rate constant, E the activation energy, R the gas constant, T the absolute temperature, and A a constant. Interpretation of the usually large effects of temperature on reaction rates (often 200300~o over a 3 ~ change in temperature) is based upon the concept of the formation of a high energy intermediate (the activated complex) which once formed, decomposes to give products and free enzyme. The rate of the overall reaction is governed primarily by the probability of forming the activated complex, which is in turn dependent upon the temperature, and the difference in Gibbs' free energy between the reactants and the activated complex (the free energy of activation, AG:~). Thus at a given temperature reaction rate is determined by the magnitude of AG:~. The influence of pressure on enzyme reaction rate, on the other hand, is dependent on the volume change occurring during activation of the enzyme* Present address: Department of Zoology, Duke University, Durham, NC 27706, U.S.A. 19
substrate complex, and can be described by the relationship: AV3~ = 2"3 R T
log kpl - log kp2 P2 - - P l
(Johnson & Eyring, 1970) where AV:~is the volume change of activation (volume of activated complex/volume of reactants), R is the gas constant (82-07 atm cma/mole °K), and kpl and kp2 velocity constants at pressures Pl and P2. It is important to appreciate that while increasing temperature will always tend to increase the kinetic energy of the reactants and hence increase reaction rate, increasing pressure may increase, decrease or have no influence on the rate depending on the relative volumes of reactants and activated complex. It should also be stressed that pressure and temperature interact, so that reaction rates will depend on the relative contributions of these parameters. When an enzyme is not functioning under saturating substrate concentrations, the effects of pressure are complicated by volume changes occurring during substrate binding. The sign and the size of these volume changes depend upon (1) the kind of weak bonding interactions involved in stabilizing enzymesubstrate complex, and (2) the relative contributions of different weak bonding interactions. An important point to bear in mind is that pressure and temperature differentially influence weak chemical bonding interactions between enzyme and ligand (Hochachka, 1974; Hochachka et al., 1975; Hochachka & Somero, 1973). In this context, it is clear that the chief functional problems faced by enzymes in abyssal organisms are (1) the maintenance of high potential reaction rates in an environment of low thermal energy and high pressure, and (2) the maintenance of weak-bond structural interactions. To gain some insight into the stra-
20
J. BALDWIN, K. B. STOREY AND P. W. HOCHACHKA
tegies adopted by abyssal organisms to maintain functional enzyme integrity at low temperatures a n d high pressures, muscle lactate dehydrogenase ( L D H M4) has been purified from the abyssal fish Antimora rostrata a n d a study made of the effects of temperature a n d pressure on reaction rates a n d weak-bond dependent properties of the enzyme. MATERIALS A N D M E T H O D S
Experimental animals Specimens of Antimora rostrata were collected with a free vehicle capture system (Phleger & Souter, 1971) at depths of about 2 km off the Kona coast of Hawaii. Skeletal muscle was removed from the animal within minutes of recovery, and stored at -20°C. Enzyme assay
Lactate dehydrogenase activity was assayed by following the oxidation of NADH at 340nm with a Gilford 2400 recording spectrophotometer or an SP1800 Unicam recording spectrophotometer incorporating a temperature controlled high pressure cell (Mustafa et al., 197l). The reaction mixture contained NADH, sodium pyruvate and 100mM sodium phosphate buffer, pH 7.5. The reaction was started by the addition of a small volume (10-50/d) of suitably diluted enzyme preparation. For the determination of kinetic parameters the enzyme was first dialysed against 100 mM sodium phosphate buffer, pH 7-5, containing 10 mM 2-mercaptoethanol. Electrophoresis
Enzyme extracts were examined by starch gel electrophoresis as described previously (Baldwin & Aleksuik, 1973), and the purity of the final preparation was checked by staining for activity and protein following acrylamide gel disc electrophoresis (Davis, 1964). Protein estimations
Protein estimations during purification were made by the method of Lowry et al. (1951) and the extinction coefficient of the final purified preparation was determined from the absorption at 205 and 280 nm, where E205 = 26"8 + (125 x A280/A205) (Holmes & Scopes, 1974). Determination of kinetic and thermodynamic parameters
Michaelis constants (Km) were determined from Woolf plots (substrate concentration/velocity vs substrate concentration), and maximum velocities (Vm,~) from Lineweaver-Burk plots (~/velocity vs ~/pyruvate concentration). The experimental energy of activation (Ea) was calculated by plotting log Vm,~ against ~/T (slope = -Ea/2.303R). Thermodynamic functions were calculated from the following relationships (Lehrer & Barker, 1970):
by centrifugation at 30,000 0 for 30 min in a Sorvall RC/2B refrigerated centrifuge. The supernatent was brought to 60% saturation by the addition of solid ammonium sulphate and the resultant precipitate collected by centrifugation, and dissolved in 20 ml of 5 mM TribHCl buffer, pH 7.6. This solution was heated at 55°C for 10min in a water bath, followed by rapid cooling on ice. Inactive precipitated material was removed by centrifugation. The supernatant from the heatstep was dialysed against 5 mM T r i b HC1 buffer, pH 7-6, and absorbed onto a DEAE cellulose (Whatman DE22) column (1.8 x 60 cm) equilibrated in the same buffer. The column was washed with 1000 ml of equilibration buffer, followed by the elution of LDH activity with a 1000ml linear gradient from 50mM to 150mM sodium chloride in 5 mM Tris-HCl buffer, pH 7.6. Fractions with the highest specific activity were combined and concentrated by membrane filtration (Amicon Diaflo, PM10). The concentrated sample was dialysed against 10raM potassium phosphate buffer, pH 5.8, and applied to a carboxymethyl-cellulose (Whatman CM 23) column (1.8 x 60cm) equilibrated with the dialysis buffer. The enzyme was eluted from the column with 10mM potassium phosphate buffer, pH 6-1. Active fractions were combined and concentrated to a volume of 20 ml by membrane filtration. Solid ammonium sulphate was added slowly with constant stirring until the first signs of turbidity appeared at about 55% saturation and the enzyme was left to crystallize at 4°C for 48 hr. The crystalline suspension of LDH was centrifuged and redissolved in 20 ml of distilled water. This crystallization procedure was repeated twice, at which stage no further increase in specific activity occurred. The purified enzyme was stored in 55y,, saturated ammonium sulphate at 4°C. RESULTS AND DISCUSSION Enzyme purification
A summary of the purification procedure is given in Table 1. The final yield from 4000 of muscle was 5 mg of L D H protein. Starch gel electrophoresis of the 30,0000 supernatent, the 60% a m m o n i u m sulphate precipitate a n d the heat treated extract gave three anodally migrating b a n d s of L D H activity. Following DEAE-cellulose c h r o m a t o g r a p h y only the most slowly migrating b a n d could be detected a n d this was assumed to be the M 4 L D H homopolymer. Acrylamide gel disc electrophoresis of the final crystallized preparation gave single coincident b a n d s of L D H activity a n d protein. A n E28 o (1 mg/ml) value of 1.87 was obtained for the purified enzyme. Table 1. Purification of Antimora LDH M4 Purification
AH~. = Ea - R T
AS:~= 4 . 5 7 6 ( 1 o g k - 10.753 - log T + 4"@6) Where AG:~ (cal/mol) is the free energy of activation, AH~ (cal/mol) the enthalpy of activation, AS~ (e.u.) the entropy of activation, T the absolute temperature, R the gas constant, Ea (cal/mol) the experimental energy of activation, and K (sec- 1) the rate constant obtained from the relationship K = Vmjmgenzyme x mol. wt (mg/m-mol) x 10-3 m-mol/#mol x 1 min/60 sec. Enzyme purification Skeletal white muscle (400g) was homogenized with 1200 ml of distilled water in a Waring blender followed
30,000
g
supernatent
60% ammonium precipitate Heat at
Step
sulphate
treatment, 55 °
10 m l n
DrAr-oe~Zu~ose ch ..... tography c:~-c~z~u~. . . . h. . . . toqraphy x3 crystallisation
Specific Activity
Yield
2
i00
24
85
190
85
452
43
565
29
1,130
25
(%)
* ~tMoles NADH/min per mg protein. Assay: 2.5mM sodium pyruvate, 0.1 mM NADH, 100 mM sodium phosphate buffer, pH 7.5.23°C and 14.7 psi.
Antimora M4 LDH activation parameters
21
Table 3. Relative specific activities (/~moles NADH/min/g muscle) of LDH and phosphorylase in skeletal white muscle of Antimora and rainbow trout Salmo 9airdneri
40
2(3
Species
Antimora Trout
LDH
Phosphorylase
37
0
817
50
E to
o x 8 E >
Assays were carried out at 25°C and 14.7 psi. The LDH reaction mixture contained 2-5 mM sodium pyruvate, 0.25 mM NADH and 0.1 M sodium phosphate buffer, pH 7.5. Phosphorylase was assayed as described by Burleigh & Schimke (1968).
6
34o
36o
1 x 105 (T)
Fig. 1. Arrhenius plot of Vma,/mg (#moles NADH/min/mg protein) vs 1/Absolute temperature for Ant#nora LDH M 4. Assay: 0.25 mM NADH, 100mM sodium phosphate buffer, pH 7.5, 14-7 psi. limax calculated from Lineweaver-Burk plots at 8 pyruvate concentrations in the range 0.1-5 mM. Effect of temperature on maximum velocity
(Wmax)
The Arrhenius plot for Antimora LDH M4 at 14.7 psi is shown in Fig. 1. The slope of the line is constant over the temperature range examined, yielding an Ea value of 13,200 _ 200 cal/mol. Catalytic efficiency Two mechanisms by which high catalytic rates can be maintained at low temperatures are the production of enzymes of high catalytic potential (low AG:~ characteristics), and increasing the amount of enzyme. Thermodynamic activation parameters for Antimora LDH M4 calculated from'the data in Fig. 1, are listed in Table 2. The AG:~ values for the Antimora enzyme are intermediate between those obtained for LDH M4 enzymes of other fish and for the homologous enzymes from mammals and birds. As a consequence the turnover number at 5°C for the Antimora enzyme is 1-3 to 6-fold higher than values for the enzyme from mammals and birds, and 1.7-3.5-fold lower than for the halibut and tuna enzyme respectively. (Baldwin, in preparation; Low et al., 1973). The high AG:~ value for the Antimora enzyme relative to these other
Table 2. Thermodynamic activation parameters for Antimora LDH M4 Assay Temperature ('c)
Turover
NO.
Ea (eal/mol)
dH~ (cal/mol)
kS = (e.u.)
~G ~ (cal/mol)
5
2.48
x
102
13200
12648
-0.25
12717
35
2.65
x
103
13200
12588
-0.39
12707
* #Moles NADH/min per mg protein. Values are given at 5 and 35°C for comparison with published data for LDH M 4 of other species (Low et al., 1973).
two fish may be explained in terms of differences in the metabolic requirements of muscle tissues in these species. The LDH M4 isoenzyme occurs in highest concentration in muscles which rely heavily on energy production from anaerobic glycolysis. On this basis, one might predict that selection for a particular catalytic efficiency of LDH M 4 will depend on both body temperature and on the level of anaerobic glycolysis required to supply energy for muscular contraction during periods of sudden rapid locomotion. The specific activity of LDH in skeletal white muscle of Antimora is about 20-fold lower than in the tissue from rainbow trout, and phosphorylase (an enzyme involved in mobilizing glycogen during anaerobic glycolysis) could not be detected (Table 3). It appears then that this tissue in Antimora is incapable of high energy output under anaerobic conditions, and this may reflect a sluggish disposition in common with other species of abyssal fish (Dreizen & Kim, 1971; Kim & Dreizen, 1971; Schutts & Isaacs, 1973). Tuna and halibut are presumably more active species than Antimora and are probably capable of achieving higher levels of anaerobic glycolysis. However, in the absence of data on tissue LDH levels in these fish, it is not possible to determine if Antimora has adjusted to a low tissue LDH level by simply selecting an enzyme with a lower turnover number, or if the amount of enzyme has also been reduced. Effect of pressure on maximum velocity (V,,o~) As the effect of pressure on the maximum velocity of enzyme catalysed reactions can vary with temperature, assays at 14.7 and 8000 psi were made at 5, 15, 25 and 30°C. The results of these experimemts are expressed in the form of an Arrhenius plot in Fig. 2. It is apparent that a pressure of 8000 psi (corresponding to a depth of about 5.4km) has no effect upon Vm,x over the temperature range 5-35°C and that the volume change of activation (AV:~) at these pressures is negligible. A similar pressure independence of Vma. at pressures up to 8000 psi has been observed for several partially purified LDH isoenzymes of abyssal Coryphenoides fishes (Moon et al., 1971). While a AV:~ value of zero is obviously compatible with enzyme function in abyssal animals, without comparable data from surface forms it is not clear if this is a specific adaptation for catalysis at high pressure, or a property of LDH enzymes in general.
22
J. BALDWIN, K. B. STOREYAND P. W. HOCHACHKA
cance and reflects a decreased affinity of the enzyme for substrate as temperature increases, thereby providing a mechanism for rate stabilization at in vivo levels of substrate (Hochachka & Somero, 1968). The observation that, at higher temperatures, pressure leads to a small reduction in the apparent Km for pyruvate is not understood in mechanistic terms. Since it is clear that carboxyl charge neutralization occurs during binding, the process should occur with a distinct volume increase (i.e. pressure should increase the Km rather than decrease it). One possibility is that the expected volume increase is not observed because of a compensating volume decrease associated with binding (Hochachka, 1975). As hydrogen bond formation is a process proceeding with a modest volume decrease, and is also thought to play a role in substrate binding to the L D H - N A D H binary complex, the observed results are consistent with the idea of a relative increase in the binding contribution of hydrogen bond based processes in the case of Antimora M 4 L D H (Hochachka, 1975). However, further studies are clearly needed to establish this point with certainty.
o A
1 .8 .6
~.4 E
@
.2
320
~0
3;30
3,'50 1 xlO 5 (T)
360
Fig. 2. Arrhenius plot of Vmax (AOD 340/min) vs l/Absolute temperature for Antimora LDH M4 at pressures of 14.7 psi (A), and 8000 psi (O). Assay conditions as for Fig. 1. Effect of temperature and pressure on enzyme-substrate affinity Of the many weak-bond dependent properties of enzymes the influence of temperature and pressure on enzyme-substrate affinity is of particular importance as it is this parameter rather than Vm,x that determines reaction rates at low in vivo substrate concentrations. The effects of temperature and pressure on the apparent Km for pyruvate are shown in Table 4. At low temperature (5°C) the apparent Km is insensitive to a pressure of 8000 psi, and of the data in Table 4, this may be the only observation of biological significance to an abyssal animal which probably never encounters habitat temperatures above 5°C. The increase in apparent Km with increasing temperature at 14.7 psi is a general property of L D H enzymes from poikilotherms (Baldwin & Aleksiuk, 1973; Hochachka & Somero, 1968; Somero, 1969). It has been proposed that this is of adaptive signifiTable 4. Effect of temperature and pressure on the apparent Km for pyruvate for Antimora LDH M 4 Assay
temperature
(°C)
Apparent
Km
Acknowledgement~This work was supported by an NRC (Canada) operating grant to PWH. R/V Alpha Helix operations were supported by the NSF (U.S.). Especial thanks are due Capt. R. Coleman and the crew of the Alpha Helix, without whose full cooperation these studies would not have been possible. REFERENCES BALDWIN J. • ALEKSUIKM. (1973) Adaptation of enzymes
to temperature: lactate and malate dehydrogenases from platypus and echidna. Comp. Biochem. Physiol. 44B, 363370. BURLEIGH G. & SCH1MKER. T. (1968) On the activities of some enzymes concerned with glycolysis and glycogenolysis in extracts of rabbit skeletal muscles. Biochem. biophys. Res. Commun. 31, 831 836. DAVIS B. J. (1964) Disc Electrophoresis--II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci., U.S. 121, 404-427. DREIZEN P. & KIM H. D. (1971) Contractile proteins of a benthic fish--I Effects of temperature and pressure on myosin ATPase. Am. Zool. II, 513-521. HOCHACHKA P. W. (1974) Temperature and pressure adaptation of the binding site of acetylcholinesterase. Biochem. J. 143, 535 539. HOCHACHKAP. W. (1975) Fitness of enzyme binding sites for their physical environment: coenzyme and substrate binding sites of M 4 LDH. Comp. Biochem. Physiol. 52B, 25-31. HOCHACHKAP. W. & SOMEROG. N. (1968) The adaptation of enzymes to temperature. Comp. Biochem. Physiol. 27, 65%668. HOCHACHKA P. W. & SOMERA G. N. (1973) Strategies of
{m~)
biochemical adaptation. W. B. Saunders, Philadelphia. HOCHACHKA P. W., STOREY K. B. & BALDWIN J. (1975)
14.7
psi
8000
psi
Design of acetylcholinesterase for its physical environment. Comp. Biochem. Physiol. 52B, 13 18.
5
0.3
0.3
15
0.6
0.3
HOLMES R. A. & SCOPES R. K. (1974) Immunochemical
25
1.0
0.4
35
2.1
1.3
homologies among vertebrate lactate dehydrogenase isozymes. Eur. J. Biochem. 43, 167 177.
Assay: 0.25 mM NADH, 100mM sodium phosphate buffer, pH 7'5. Apparent K m values were determined from Woolf plots (pyruvate concentration/velocity vs pyruvate concentration) at 0-8 pyruvate concentrations over the range 0.1-5 mM).
JOHNSON F. A. & EYRING H. (1970) The kinetic basis of
pressure effects in biology and chemistry. In High Pressure Effects on Cellular Processes (Edited by ZIMMERMAN A. M.), pp. 1-44. KIM H. D. & DREIZEN P. (1971) Contractile proteins of a benthic f i s ~ I I . Composition and ATPase properties of Actomyosin. Am. Zool. l l , 525-529.
Antimora M 4 LDH activation parameters
LEHRER G. M. & BARKER R. (1970) Conformational changes in rabbit muscle aldolase. Kinetic studies. Biochem. J. 9, 1533-1539. Low P. S., BADA J. L. & SOMEROG. M. (1973) Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation. Proc. natn Acad. Sci., U.S.A. 70, 430-432. LOWRY O. H., ROSENBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MOON T. W., MUSTAFA T. & HOCHACHKA P. W. (1971) Effects of hydrostatic pressure on catalysis by different lactate dehydrogenase isoenzymes from tissues of an abyssal fish. Am. Zool. 11, 473-478.
23
MUSTAFA T., MOON T. W. & HOCHACHKA P. W. (1971) Effects of pressure and temperature on the catalytic and regulatory properties of muscle pyruvate kinase from an off-shore benthic fish. Am. Zool. 11, 451-466. PHLEGER C. F. 8/. SOUTARA. (1971) Free vehicles and deep sea biology. Am. Zool. 11,409-418. SOMERO G. N. (1969) Enzymic mechanisms of temperature compensation: immediate and evolutionary effects of temperature on enzymes of aquatic poikilotherms. Am. Zool. 103, 517-530. SHUTTS L. J. & ISAACS J. D. (1973) Life on the abyssal floor. 16 mm Film, Scripps Inst. Oceanography, La JoUa, California.
LACTATE D E H Y D R O G E N A S E M4 OF AN ABYSSAL FISH" STRATEGIES FOR F U N C T I O N AT LOW TEMPERATURE A N D HIGH PRESSURE JOHN BALDWINt, K. B. STOREY2. AND P. W. HOCHACHKA2 t Genetics Department, Research School of Biological Sciences, The Australian National University, Canberra, Australia 2 Department of Zoology, University of British Columbia, Vancouver, B.C., V6T 1WS, Canada (Received 11 October 1974)
Abstraet--l. Lactate dehydrogenase M 4 purified from the abyssal fish Antimora rostrata displays lower AG:~ characteristics and a higher turnover number than the homologous enzyme of homeotherms. 2. Pressures of 8000psi have no effect upon Vm~x, and the volume change of activation (AV:~)between 14.7 and 8000psi is negligible. 3. The apparent Km of the enzyme for pyruvate is insensitive to pressure at low temperature. 4. These properties are clearly compatible with function at the low temperature and high pressure environment of the abyssal floor.
INTRODUCTION
Low TEMPERATUREand high pressure are two environmental parameters which exert a direct influence upon enzyme function in deep sea fish. The effect of temperature and pressure on enzyme catalysis can be conveniently divided into two classes: (1) rate effects, which determine how rapidly equilibrium is attained between substrates and products; (2) weak-bond structural effects, which influence those properties of the enzyme that depend upon non-covalent bonding. Examples of this second class are 3° and 4 ° structure and the binding of substrates and modulator molecules. The effect of temperature on enzyme reaction rates is described by the Arrhenius equation: k
=
Ae -E/RT
where k is a rate constant, E the activation energy, R the gas constant, T the absolute temperature, and A a constant. Interpretation of the usually large effects of temperature on reaction rates (often 200300~o over a 3 ~ change in temperature) is based upon the concept of the formation of a high energy intermediate (the activated complex) which once formed, decomposes to give products and free enzyme. The rate of the overall reaction is governed primarily by the probability of forming the activated complex, which is in turn dependent upon the temperature, and the difference in Gibbs' free energy between the reactants and the activated complex (the free energy of activation, AG:~). Thus at a given temperature reaction rate is determined by the magnitude of AG:~. The influence of pressure on enzyme reaction rate, on the other hand, is dependent on the volume change occurring during activation of the enzyme* Present address: Department of Zoology, Duke University, Durham, NC 27706, U.S.A. 19
substrate complex, and can be described by the relationship: AV3~ = 2"3 R T
log kpl - log kp2 P2 - - P l
(Johnson & Eyring, 1970) where AV:~is the volume change of activation (volume of activated complex/volume of reactants), R is the gas constant (82-07 atm cma/mole °K), and kpl and kp2 velocity constants at pressures Pl and P2. It is important to appreciate that while increasing temperature will always tend to increase the kinetic energy of the reactants and hence increase reaction rate, increasing pressure may increase, decrease or have no influence on the rate depending on the relative volumes of reactants and activated complex. It should also be stressed that pressure and temperature interact, so that reaction rates will depend on the relative contributions of these parameters. When an enzyme is not functioning under saturating substrate concentrations, the effects of pressure are complicated by volume changes occurring during substrate binding. The sign and the size of these volume changes depend upon (1) the kind of weak bonding interactions involved in stabilizing enzymesubstrate complex, and (2) the relative contributions of different weak bonding interactions. An important point to bear in mind is that pressure and temperature differentially influence weak chemical bonding interactions between enzyme and ligand (Hochachka, 1974; Hochachka et al., 1975; Hochachka & Somero, 1973). In this context, it is clear that the chief functional problems faced by enzymes in abyssal organisms are (1) the maintenance of high potential reaction rates in an environment of low thermal energy and high pressure, and (2) the maintenance of weak-bond structural interactions. To gain some insight into the stra-
20
J. BALDWIN, K. B. STOREY AND P. W. HOCHACHKA
tegies adopted by abyssal organisms to maintain functional enzyme integrity at low temperatures a n d high pressures, muscle lactate dehydrogenase ( L D H M4) has been purified from the abyssal fish Antimora rostrata a n d a study made of the effects of temperature a n d pressure on reaction rates a n d weak-bond dependent properties of the enzyme. MATERIALS A N D M E T H O D S
Experimental animals Specimens of Antimora rostrata were collected with a free vehicle capture system (Phleger & Souter, 1971) at depths of about 2 km off the Kona coast of Hawaii. Skeletal muscle was removed from the animal within minutes of recovery, and stored at -20°C. Enzyme assay
Lactate dehydrogenase activity was assayed by following the oxidation of NADH at 340nm with a Gilford 2400 recording spectrophotometer or an SP1800 Unicam recording spectrophotometer incorporating a temperature controlled high pressure cell (Mustafa et al., 197l). The reaction mixture contained NADH, sodium pyruvate and 100mM sodium phosphate buffer, pH 7.5. The reaction was started by the addition of a small volume (10-50/d) of suitably diluted enzyme preparation. For the determination of kinetic parameters the enzyme was first dialysed against 100 mM sodium phosphate buffer, pH 7-5, containing 10 mM 2-mercaptoethanol. Electrophoresis
Enzyme extracts were examined by starch gel electrophoresis as described previously (Baldwin & Aleksuik, 1973), and the purity of the final preparation was checked by staining for activity and protein following acrylamide gel disc electrophoresis (Davis, 1964). Protein estimations
Protein estimations during purification were made by the method of Lowry et al. (1951) and the extinction coefficient of the final purified preparation was determined from the absorption at 205 and 280 nm, where E205 = 26"8 + (125 x A280/A205) (Holmes & Scopes, 1974). Determination of kinetic and thermodynamic parameters
Michaelis constants (Km) were determined from Woolf plots (substrate concentration/velocity vs substrate concentration), and maximum velocities (Vm,~) from Lineweaver-Burk plots (~/velocity vs ~/pyruvate concentration). The experimental energy of activation (Ea) was calculated by plotting log Vm,~ against ~/T (slope = -Ea/2.303R). Thermodynamic functions were calculated from the following relationships (Lehrer & Barker, 1970):
by centrifugation at 30,000 0 for 30 min in a Sorvall RC/2B refrigerated centrifuge. The supernatent was brought to 60% saturation by the addition of solid ammonium sulphate and the resultant precipitate collected by centrifugation, and dissolved in 20 ml of 5 mM TribHCl buffer, pH 7.6. This solution was heated at 55°C for 10min in a water bath, followed by rapid cooling on ice. Inactive precipitated material was removed by centrifugation. The supernatant from the heatstep was dialysed against 5 mM T r i b HC1 buffer, pH 7-6, and absorbed onto a DEAE cellulose (Whatman DE22) column (1.8 x 60 cm) equilibrated in the same buffer. The column was washed with 1000 ml of equilibration buffer, followed by the elution of LDH activity with a 1000ml linear gradient from 50mM to 150mM sodium chloride in 5 mM Tris-HCl buffer, pH 7.6. Fractions with the highest specific activity were combined and concentrated by membrane filtration (Amicon Diaflo, PM10). The concentrated sample was dialysed against 10raM potassium phosphate buffer, pH 5.8, and applied to a carboxymethyl-cellulose (Whatman CM 23) column (1.8 x 60cm) equilibrated with the dialysis buffer. The enzyme was eluted from the column with 10mM potassium phosphate buffer, pH 6-1. Active fractions were combined and concentrated to a volume of 20 ml by membrane filtration. Solid ammonium sulphate was added slowly with constant stirring until the first signs of turbidity appeared at about 55% saturation and the enzyme was left to crystallize at 4°C for 48 hr. The crystalline suspension of LDH was centrifuged and redissolved in 20 ml of distilled water. This crystallization procedure was repeated twice, at which stage no further increase in specific activity occurred. The purified enzyme was stored in 55y,, saturated ammonium sulphate at 4°C. RESULTS AND DISCUSSION Enzyme purification
A summary of the purification procedure is given in Table 1. The final yield from 4000 of muscle was 5 mg of L D H protein. Starch gel electrophoresis of the 30,0000 supernatent, the 60% a m m o n i u m sulphate precipitate a n d the heat treated extract gave three anodally migrating b a n d s of L D H activity. Following DEAE-cellulose c h r o m a t o g r a p h y only the most slowly migrating b a n d could be detected a n d this was assumed to be the M 4 L D H homopolymer. Acrylamide gel disc electrophoresis of the final crystallized preparation gave single coincident b a n d s of L D H activity a n d protein. A n E28 o (1 mg/ml) value of 1.87 was obtained for the purified enzyme. Table 1. Purification of Antimora LDH M4 Purification
AH~. = Ea - R T
AS:~= 4 . 5 7 6 ( 1 o g k - 10.753 - log T + 4"@6) Where AG:~ (cal/mol) is the free energy of activation, AH~ (cal/mol) the enthalpy of activation, AS~ (e.u.) the entropy of activation, T the absolute temperature, R the gas constant, Ea (cal/mol) the experimental energy of activation, and K (sec- 1) the rate constant obtained from the relationship K = Vmjmgenzyme x mol. wt (mg/m-mol) x 10-3 m-mol/#mol x 1 min/60 sec. Enzyme purification Skeletal white muscle (400g) was homogenized with 1200 ml of distilled water in a Waring blender followed
30,000
g
supernatent
60% ammonium precipitate Heat at
Step
sulphate
treatment, 55 °
10 m l n
DrAr-oe~Zu~ose ch ..... tography c:~-c~z~u~. . . . h. . . . toqraphy x3 crystallisation
Specific Activity
Yield
2
i00
24
85
190
85
452
43
565
29
1,130
25
(%)
* ~tMoles NADH/min per mg protein. Assay: 2.5mM sodium pyruvate, 0.1 mM NADH, 100 mM sodium phosphate buffer, pH 7.5.23°C and 14.7 psi.
Antimora M4 LDH activation parameters
21
Table 3. Relative specific activities (/~moles NADH/min/g muscle) of LDH and phosphorylase in skeletal white muscle of Antimora and rainbow trout Salmo 9airdneri
40
2(3
Species
Antimora Trout
LDH
Phosphorylase
37
0
817
50
E to
o x 8 E >
Assays were carried out at 25°C and 14.7 psi. The LDH reaction mixture contained 2-5 mM sodium pyruvate, 0.25 mM NADH and 0.1 M sodium phosphate buffer, pH 7.5. Phosphorylase was assayed as described by Burleigh & Schimke (1968).
6
34o
36o
1 x 105 (T)
Fig. 1. Arrhenius plot of Vma,/mg (#moles NADH/min/mg protein) vs 1/Absolute temperature for Ant#nora LDH M 4. Assay: 0.25 mM NADH, 100mM sodium phosphate buffer, pH 7.5, 14-7 psi. limax calculated from Lineweaver-Burk plots at 8 pyruvate concentrations in the range 0.1-5 mM. Effect of temperature on maximum velocity
(Wmax)
The Arrhenius plot for Antimora LDH M4 at 14.7 psi is shown in Fig. 1. The slope of the line is constant over the temperature range examined, yielding an Ea value of 13,200 _ 200 cal/mol. Catalytic efficiency Two mechanisms by which high catalytic rates can be maintained at low temperatures are the production of enzymes of high catalytic potential (low AG:~ characteristics), and increasing the amount of enzyme. Thermodynamic activation parameters for Antimora LDH M4 calculated from'the data in Fig. 1, are listed in Table 2. The AG:~ values for the Antimora enzyme are intermediate between those obtained for LDH M4 enzymes of other fish and for the homologous enzymes from mammals and birds. As a consequence the turnover number at 5°C for the Antimora enzyme is 1-3 to 6-fold higher than values for the enzyme from mammals and birds, and 1.7-3.5-fold lower than for the halibut and tuna enzyme respectively. (Baldwin, in preparation; Low et al., 1973). The high AG:~ value for the Antimora enzyme relative to these other
Table 2. Thermodynamic activation parameters for Antimora LDH M4 Assay Temperature ('c)
Turover
NO.
Ea (eal/mol)
dH~ (cal/mol)
kS = (e.u.)
~G ~ (cal/mol)
5
2.48
x
102
13200
12648
-0.25
12717
35
2.65
x
103
13200
12588
-0.39
12707
* #Moles NADH/min per mg protein. Values are given at 5 and 35°C for comparison with published data for LDH M 4 of other species (Low et al., 1973).
two fish may be explained in terms of differences in the metabolic requirements of muscle tissues in these species. The LDH M4 isoenzyme occurs in highest concentration in muscles which rely heavily on energy production from anaerobic glycolysis. On this basis, one might predict that selection for a particular catalytic efficiency of LDH M 4 will depend on both body temperature and on the level of anaerobic glycolysis required to supply energy for muscular contraction during periods of sudden rapid locomotion. The specific activity of LDH in skeletal white muscle of Antimora is about 20-fold lower than in the tissue from rainbow trout, and phosphorylase (an enzyme involved in mobilizing glycogen during anaerobic glycolysis) could not be detected (Table 3). It appears then that this tissue in Antimora is incapable of high energy output under anaerobic conditions, and this may reflect a sluggish disposition in common with other species of abyssal fish (Dreizen & Kim, 1971; Kim & Dreizen, 1971; Schutts & Isaacs, 1973). Tuna and halibut are presumably more active species than Antimora and are probably capable of achieving higher levels of anaerobic glycolysis. However, in the absence of data on tissue LDH levels in these fish, it is not possible to determine if Antimora has adjusted to a low tissue LDH level by simply selecting an enzyme with a lower turnover number, or if the amount of enzyme has also been reduced. Effect of pressure on maximum velocity (V,,o~) As the effect of pressure on the maximum velocity of enzyme catalysed reactions can vary with temperature, assays at 14.7 and 8000 psi were made at 5, 15, 25 and 30°C. The results of these experimemts are expressed in the form of an Arrhenius plot in Fig. 2. It is apparent that a pressure of 8000 psi (corresponding to a depth of about 5.4km) has no effect upon Vm,x over the temperature range 5-35°C and that the volume change of activation (AV:~) at these pressures is negligible. A similar pressure independence of Vma. at pressures up to 8000 psi has been observed for several partially purified LDH isoenzymes of abyssal Coryphenoides fishes (Moon et al., 1971). While a AV:~ value of zero is obviously compatible with enzyme function in abyssal animals, without comparable data from surface forms it is not clear if this is a specific adaptation for catalysis at high pressure, or a property of LDH enzymes in general.
22
J. BALDWIN, K. B. STOREYAND P. W. HOCHACHKA
cance and reflects a decreased affinity of the enzyme for substrate as temperature increases, thereby providing a mechanism for rate stabilization at in vivo levels of substrate (Hochachka & Somero, 1968). The observation that, at higher temperatures, pressure leads to a small reduction in the apparent Km for pyruvate is not understood in mechanistic terms. Since it is clear that carboxyl charge neutralization occurs during binding, the process should occur with a distinct volume increase (i.e. pressure should increase the Km rather than decrease it). One possibility is that the expected volume increase is not observed because of a compensating volume decrease associated with binding (Hochachka, 1975). As hydrogen bond formation is a process proceeding with a modest volume decrease, and is also thought to play a role in substrate binding to the L D H - N A D H binary complex, the observed results are consistent with the idea of a relative increase in the binding contribution of hydrogen bond based processes in the case of Antimora M 4 L D H (Hochachka, 1975). However, further studies are clearly needed to establish this point with certainty.
o A
1 .8 .6
~.4 E
@
.2
320
~0
3;30
3,'50 1 xlO 5 (T)
360
Fig. 2. Arrhenius plot of Vmax (AOD 340/min) vs l/Absolute temperature for Antimora LDH M4 at pressures of 14.7 psi (A), and 8000 psi (O). Assay conditions as for Fig. 1. Effect of temperature and pressure on enzyme-substrate affinity Of the many weak-bond dependent properties of enzymes the influence of temperature and pressure on enzyme-substrate affinity is of particular importance as it is this parameter rather than Vm,x that determines reaction rates at low in vivo substrate concentrations. The effects of temperature and pressure on the apparent Km for pyruvate are shown in Table 4. At low temperature (5°C) the apparent Km is insensitive to a pressure of 8000 psi, and of the data in Table 4, this may be the only observation of biological significance to an abyssal animal which probably never encounters habitat temperatures above 5°C. The increase in apparent Km with increasing temperature at 14.7 psi is a general property of L D H enzymes from poikilotherms (Baldwin & Aleksiuk, 1973; Hochachka & Somero, 1968; Somero, 1969). It has been proposed that this is of adaptive signifiTable 4. Effect of temperature and pressure on the apparent Km for pyruvate for Antimora LDH M 4 Assay
temperature
(°C)
Apparent
Km
Acknowledgement~This work was supported by an NRC (Canada) operating grant to PWH. R/V Alpha Helix operations were supported by the NSF (U.S.). Especial thanks are due Capt. R. Coleman and the crew of the Alpha Helix, without whose full cooperation these studies would not have been possible. REFERENCES BALDWIN J. • ALEKSUIKM. (1973) Adaptation of enzymes
to temperature: lactate and malate dehydrogenases from platypus and echidna. Comp. Biochem. Physiol. 44B, 363370. BURLEIGH G. & SCH1MKER. T. (1968) On the activities of some enzymes concerned with glycolysis and glycogenolysis in extracts of rabbit skeletal muscles. Biochem. biophys. Res. Commun. 31, 831 836. DAVIS B. J. (1964) Disc Electrophoresis--II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci., U.S. 121, 404-427. DREIZEN P. & KIM H. D. (1971) Contractile proteins of a benthic fish--I Effects of temperature and pressure on myosin ATPase. Am. Zool. II, 513-521. HOCHACHKA P. W. (1974) Temperature and pressure adaptation of the binding site of acetylcholinesterase. Biochem. J. 143, 535 539. HOCHACHKAP. W. (1975) Fitness of enzyme binding sites for their physical environment: coenzyme and substrate binding sites of M 4 LDH. Comp. Biochem. Physiol. 52B, 25-31. HOCHACHKAP. W. & SOMEROG. N. (1968) The adaptation of enzymes to temperature. Comp. Biochem. Physiol. 27, 65%668. HOCHACHKA P. W. & SOMERA G. N. (1973) Strategies of
{m~)
biochemical adaptation. W. B. Saunders, Philadelphia. HOCHACHKA P. W., STOREY K. B. & BALDWIN J. (1975)
14.7
psi
8000
psi
Design of acetylcholinesterase for its physical environment. Comp. Biochem. Physiol. 52B, 13 18.
5
0.3
0.3
15
0.6
0.3
HOLMES R. A. & SCOPES R. K. (1974) Immunochemical
25
1.0
0.4
35
2.1
1.3
homologies among vertebrate lactate dehydrogenase isozymes. Eur. J. Biochem. 43, 167 177.
Assay: 0.25 mM NADH, 100mM sodium phosphate buffer, pH 7'5. Apparent K m values were determined from Woolf plots (pyruvate concentration/velocity vs pyruvate concentration) at 0-8 pyruvate concentrations over the range 0.1-5 mM).
JOHNSON F. A. & EYRING H. (1970) The kinetic basis of
pressure effects in biology and chemistry. In High Pressure Effects on Cellular Processes (Edited by ZIMMERMAN A. M.), pp. 1-44. KIM H. D. & DREIZEN P. (1971) Contractile proteins of a benthic f i s ~ I I . Composition and ATPase properties of Actomyosin. Am. Zool. l l , 525-529.
Antimora M 4 LDH activation parameters
LEHRER G. M. & BARKER R. (1970) Conformational changes in rabbit muscle aldolase. Kinetic studies. Biochem. J. 9, 1533-1539. Low P. S., BADA J. L. & SOMEROG. M. (1973) Temperature adaptation of enzymes: roles of the free energy, the enthalpy, and the entropy of activation. Proc. natn Acad. Sci., U.S.A. 70, 430-432. LOWRY O. H., ROSENBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. MOON T. W., MUSTAFA T. & HOCHACHKA P. W. (1971) Effects of hydrostatic pressure on catalysis by different lactate dehydrogenase isoenzymes from tissues of an abyssal fish. Am. Zool. 11, 473-478.
23
MUSTAFA T., MOON T. W. & HOCHACHKA P. W. (1971) Effects of pressure and temperature on the catalytic and regulatory properties of muscle pyruvate kinase from an off-shore benthic fish. Am. Zool. 11, 451-466. PHLEGER C. F. 8/. SOUTARA. (1971) Free vehicles and deep sea biology. Am. Zool. 11,409-418. SOMERO G. N. (1969) Enzymic mechanisms of temperature compensation: immediate and evolutionary effects of temperature on enzymes of aquatic poikilotherms. Am. Zool. 103, 517-530. SHUTTS L. J. & ISAACS J. D. (1973) Life on the abyssal floor. 16 mm Film, Scripps Inst. Oceanography, La JoUa, California.
