Comp. Biochem. Physiol., 1978, Vol. 59A, pp. 31 to 36. Peroamon Press. Printed in Great Britain

TEMPERATURE AND pH EFFECTS ON CATALYTIC PROPERTIES OF LACTATE DEHYDROGENASE FROM

PELAGIC FISH

ALDIS VALKIRS

The Lockheed Center for Marine Research, Carlsbad, CA 92008, U.S.A.

(Received 11 March 1977)

AImmet--1. Enzyme-substrate affinity (Kin) in some pelagic fish tissues is profoundly influenced by temperature and pH at 10w substrate concentrations. Decreasing temperature causes an increase in enzyme-substrate affinity, and hence tends to compensate for decreasing thermal energy. Such an effect has been found in fish which routinely experience large temperature changes daily, as well as in fish which exist in a more static thermal regime.

2. Values of Qlo were found to be near 1 at low substrate levels. Under such conditions the reaction rate is not largely effected by temperature but becomes more sensitive to variations in Kin.

3. Arrhenius plots of log Vm., VS I/T also indicate decreasing effects of temperature over reaction rate at low substrate concentrations. The slopes of the Arrhenius plots exhibit a tendency to decrease with lower substrate levels.

INTRODUCTION

The effect of temperature on cnzymc-substrate (E-S) affinity measured at physiological substrate concen- trations has received increasing attention in recent years, and has been reviewed by Hochachka (1967); Hochachka & Somero (1971, 1973); and by Fry & Hochachka (1970). Hochachka & Somero (1968) found that lactate dehydrogenase (LDH) affinity for pyruvate varies with temperature and approaches a maximum value within a poikilothermic organism's normal temperature range. Baldwin & Hochachka (1970) also noted this effect with acetylcholinesterasc in trout. Temperature may act as a positive modu- lator by increasing E-S affinity when substrate con- centrations are low (Atkinson, 1966; Somero, 1972).

E-S affinity may vary inversely with temperature over a large portion of a species' thermal habitat. An increase in E-S affinity accompanying a decrease in temperature will partially, or fully, offset the loss in catalytic velocity resulting from decreased kinetic energy. This effect serves to stabilize the reaction over a wide range of temperatures (Somero & Hochachka, 1969; Somero, 1969).

At higher temperatures, the apparent Km increases as the temperature is raised, thus keeping the reaction velocity constant as more thermal energy is made available. Such a compensating effect was also noted for citrate synthasc from trout (Hochachka & Lewis, 1970), and for isocitrate dehydrogenase from trout (Moon & Hochachka, 1971).

This plasticity in control over reaction velocity by varying the apparent Km is also noted in several other poikilotherms as well as in many fish species. Gerez De Burgos et al. (1973) studied the effects of temperature and pH on the kinetics of LDH isoen- zymes from a snake (Bothrops neuwiedii) and from beef heart. The apparent Km values for poikilotherm LDH isoenzymes were clearly reduced at lower energy assay temperatures, indicating compensation

for reduced thermal energy. Apparent Km values for beef isoenzymes were slightly lower at reduced tem- peratures, but to a lesser extent than snake isozymes. At any given temperature (10-35°(2) the apparent Krn increased with increasing pH. Snake LDH isoenzymes were found to be more sensitive to pH changes than were bovine LDH isozymes. Hoskins & Aleksiuk (1973) noted a "reduction in Km with decreasing tem- perature with malate dehydrogenase from another reptile, Thamnophis sirtalis. Baldwin & Aleksiuk (1973) obtained similar results with lactate and malate dehydrogenases from platypus and echidna. Hochachka & Lewis (1971) noted that changes in pH may strongly effect Kin-temperature relationships in fish tissue LDH. At low pH values, the absolute value of the Km was found to be least temperature sensitive, while at slightly alkaline pH values (above pH 7.5), the Km increased distinctly with increasing tempera- ture. Studies by Rahn (1965) and Reeves & Wilson (1969) showed that blood, and intracellular pH of some poikilotherms increased as temperature de- creased.

This present study was undertaken to examine the effects of temperature and pH on the kinetics of LDH from open ocean fish. It is well known that certain groups of fish migrate diurnally from depths they occupy during daylight toward the surface at night (Tucker, 1951; Marshall, 1951, 1960; Barham; 1957)~ During such a migration, which commonly spans 3(gl-400 m, environmental temperatures may vary by 20°C. Such fish provide an excellent source for study- ing effects of temperature and pH on enzyme-sub- strate affinity and its possible application to imme- diate rate compensation of enzyme reactions.

White muscle tissue, which typically has a high rate of glycolytic activity, (Hochachka, 1973; Hocliachka & Somero, 1973) was used for this study. A compari- son is made between the kinetic behavior of LDH from diurnally migrating fish and flying fish. Hying fish are strictly surface dwelling, and are not exposed

31

32 A L D I S V A L K I R S

to the thermal differences encountered by diurnal migrators, but may frequently rely on glycolytic meta- bolism to accomplish their unique form of movement.

MATERIALS AND METHODS

Experimental animals

Collections were made in the eastern tropical Pacific Ocean aboard the USNS De Steiguer during the month of September, 1974. Flying fish (Exocoetus volitans) were collected at night in surface water by dip net at 16°55.5'N; 118°40.5'W. Mesopelagie fish of the family Myctophidae (Hygophum atratum) were collected at night in surface waters with a surface skimming net at 17°25'N; l18°W and at 17°13.5'N; 118°01'W. Tissues from living specimens were excised, washed in cold 0.1 M potassium phosphate buffer pH 7.39, and frozen immediately over dry ice.

Frozen tissue was stored in a deep freeze aboard ship at -20°C and subsequently at -20°C in the laboratory. Fishes from which tissue samples had been taken were labelled, preserved and kept for positive identification.

Assay method Tissue samples were placed in 0.03 M potassium phos-

phate buffer, pH 7.4, homogenized in a mechanized tissue grinder and emulsified in a sonicator-cell disi-uptor. Homo- genates were centrifuged for 20 min at 20,000 g in a Sorvall RC-2B refrigerated centrifuge at 2°C. The resulting super- natant fractions were made 1% with bovine serum albumin and used directly for measurement of LDH activity. LDH activity was measured by observing the oxidation of reduced nicotinamide adenine dinucleotide (NADH) at 340nm in a Perkin-Elmer 124D dual beam spectrophot- ometer equipped with an SRG recorder. Appropriate dilu- tions of the enzyme solution were made in order that the change in absorbance remained linear for at least 1 min. A Fisher Isotherm constant temperature bath was used to maintain cuvette temperatures at any level between 0 and 50°C. Reaction mixtures contained 5 × 10-SM NADH and varying levels of pyruvate aad LDH in 3.0 ml of 0.03M potassium phosphate buffer (10mm light path cuvette). The enzyme fraction was added last after the reac- tion mixture had been thoroughly mixed and brought to the desired assay temperature. Care was taken to make up all buffers so the pH would be accurate at the respective experimental temperatures of the assay. This was accom- plished with a thermally equilibrated pH electrode (Zirino, 1975) connected to the constant temperature bath.

Values of Km were calculated by the method of Line- weaver and Burk using double reciprocal plots of velocity versus substrate concentration. The average of two assays was used to determine the reaction velocity. Single tissue samples of flying fish white muscle were used to determine LDH activity. LDH activity in H. atratum white muscle was determined from tissue samples pooled from five speci- mens. Sodium pyruvate, grade A, was purchased from Sigma, St. Louis. NADH was purchased from Calbiochem, San Diego.

RESULTS

Temperature and pH effects on Km

Lactate dehydrogenase (LDH) from myctophid (Hygophura atratum) white muscle showed positive thermal modulation, meaning the apparent Km varied directly with temperature. Hence, as temperature de- creased the enzyme-substrate affinity increased (Fig. 1). This relationship occurred at the three pH's tested. There was also an increase in Km values with increas- ing pH at any given temperature. The Km was most

7 0 - -

~× 6o- / ~; 5o--

e

3 0 - - •

A ~

I I I I 5 J5 25 35

T e m p e r o t u r e , °C

Fig. 1. Effect of temperature and pH on Km (pyruvate) of myctophid (H. atratum) white muscle LDH. Conditions of assay as indicated in Methods. • = pH 7.0; • = pH

7.4; • = pH 8.0.

temperature sensitive at the most alkaline pH tested (pn = 8.0).

Flying fish (Exocoetus volitans) white muscle LDH demonstrated positive thermal modulation at all pH values and thermal intervals with one exception (Fig. 2). At pH 8.0 between 25 and 35°C, the Km increased as temperature decreased. An attempt was made to duplicate the Km data at pH 8.0 and 7.0 at 25 and 35°C with white muscle tissue from another E. roll- tans specimen (specimen No. 2). These results are shown in Fig. 3.

The effect of increasing temperature (from 25 to 35°C) at pH 8.0 showed an increase i.ta Km for this tissue specimen (Fig. 3). At pH 7.0 the Km increased over this same temperature interval; however, the

1 0 - -

9 - -

8 - -

7 - -

x

5--

4 - -

3 - -

2 - -

I - -

I I I I 5 15 25 35

T e m p e r o t u r e , °C

Fig. 2. Effect of temperature and pH on Km (pyruvate) of flying fish (E. volitans) specimen No. 1 white muscle LDH. Conditions of assay as indicated in Methods.

• = p H 7.0; • = pH 7.4; • = pH 8.0.

Temperature and pH effects on catalytic properties 33

7

~ 6

5

4

I I I I 5 f5 25 35

Tempeto~'ure, *C

Fig. 3. Effect of temperature and pH on Km (pyruvate) of flying fish (E. mlitans) specimen No. 2 white muscle LDH. Conditions of assay as indicated in Methods.

• = p H 7.0; • = 7.4; • = p H 8.0.

magnitude of the Km was different in the two flying fish specimens. A single assay was performed at pH 7.4 (25°C) and this Km value was also different from results obtained with tissue from the first flying fish specimen.

Temperature coefficients (Qlo)

The Qlo of myctophid (H. atratum) white muscle LDH showed varying effects of substrate concen- tration. The data for H. atratum white muscle LDH is shown in Table 1. When Qlo was calculated between 25 and 35°C, the values generally increased with increasing substrate concentration and decreased with increasing pH. When Qlo values for H. atratura white muscle LDH were calculated I~¢tween 15 and 25°C, an increase in Qlo with increasing substrate concentrations was again seen. A decrease in Qlo values with increasing pH was also evident. H. atra- mm white muscle LDH showed increased values of Qlo with increasing substrate concentrations at pH 7.0 between 5 and 15°C {Table 1l

Table 1. Effect of pyruvate concentration and pH on the temperature coefficient (Qlo) for myctophid (H. atratum)

white muscle LDH

Pyruvate c o n c l l

(raM) pH 7.0 pH 7.4 pH 8.0

Qlo between 25 and 35°C 0.033 0.77 0.84 0.56 0.33 0.98 0.93 0.63 1.66 1.44 1.23 0.76 3.3 1.59 1.43 1.02

Qlo between 15 and 25°C 0.033 2.54 2.18 1.78 0.33 3.05 2.30 1.78 1.66 3.62 3.37 2.96 3.3 3.93 3.53 2.85

Q:o between 5 and 15°C 0.033 1.71 0.33 1.95 1.66 2.35

Table 2. Effect of pyruvate concentration and pH on the temperature coefficient (Qlo) for flying fish (E. volitans) specimen No. 1 white muscle LDH. (Data is taken from

the same specimen represented in Fig. 2)

Pyruvate conch ( m M ) pH 7.0 pH 7.4 pH 8.0

Qto between 25 and 35°C 0.019 0.92 0.88 0.88 0.067 0.87 0.82 0.65 0.19 1.97 1.00 0.66 3.3 1.00 0.95 0.65

O:0 between 15 and 25°C 0.019 1.04 1.23 1.00 0.067 1.22 1.16 1.35 0.19 1.20 1.25 1.17 3.3 1.86 1.38 1.35

Qlo between 5 and 15°C 0.019 1.18 0.067 1.34 0.19 1.60 3.3 1.77

Table 3. Effect of pyruvate concentration and pH on the temperature coefficient (Q:0) for fly- ing fish (E. volitans) specimen No. 2 white muscle LDH. (Data is taken from the specimen

represented in Fig. 3)

Pyruvate COfiCn

(raM) pH 7.0 pH 8.0

Q,o between 25 and 35°C 0.033 0.83 0.67 - 0.33 0.93 0.67 1.66 1.55 0.86

- 2 O0

-I 5 0

>

~ -I O0

- 0 5 0

I - I I I 360 347 336 325

I / T X l O 5, *K

Fig. 4. Arrhenius plot of LDH activity for myctophid (H. atrotum) white muscle tissue. Activity was determined at 3.3 mM pyruvate, &; 0.33 mM pyruvate, • ; and 0.033 mM pyruvate, I . Reaction mixtures contained 0.05 mM NADH

and 0.03 M potassium phosphate buffer, pH 7.0.

34 ALDIS VALKIRS

-I 50 --

E

~ -10(3 --

-050 --

I I I I 36O 347 336 325

I/Tx IO 5, OK

Fig. 5. Arrhenius plot of LDH activity for flying fish (E. oolitans) white muscle tissue.-,Acti~y was determined at 9.6 mM pyruvate, A; 0.19 mM pyruvate, O; and 0.067 mM pyruvate, I Reaction mixtures contained 0.05-ram NADH

and 0.03 M potassium phosphate buffer, pH 7.4.

Values of Qlo calculated for flying fish (E. volitans) white muscle LDH (Tables 2 and 3) were low. In- creases in Qt0 with increasing substrate concen- trations were not always seen with the flying fish enzyme. The Qto values calculated for white muscle LDH of the two flying fish over the same thermal interval, pH, and at similar substrate concentrations were of the same magnitude (Tables 2 and 3).

Arrhenius plots

Arrhenius plots of log Vmx vs I /T (K) exhibited a tendency to decrease in slope and hence in activation energy, with decreasing substrate concentration for LDH from all tissues assayed (Figs 4 and 5). At pyru- vate concentrations of 10-~-10 -5 ~1, which may be considered physiological (Freed, 1971; Mayerle & Butler, 1971), the slopes of the Arrhenius plots became flat, or were slightly negative over certain thermal intervals (Figs 4 and 5). Under these condi- tions the Q~o may be 1 or less. This data is presented in Tables 1-3 where Qto values were I or less between 25 and 35°C at the respective substrate concentrations and pH values indicated in Figs 4 and 5.

D I S C U S S I O N

Myctophid white muscle LDH

Myctophid fish routinely migrate vertically in the water column as much as several hundred meters dur- ing a diurnal cycle (Alexander, 19701 In executing such a migration, these fish may encounter extreme differences in temperature. Water temperatures were 26.8°C at the surface and 8°C at 450 m in the area where specimens were collected. It has been demon- strated by Rahn (1965), that reduction in environmen- tal temperature results in an increase in blood pH of some fishes, The increase amounts to 0.014--0.01 pH units/°C. Reeves & Wilson (1969) have shown that such an effect on pH is also true in the intracellular environment of some poikilotherms.

Changes in both temperature and pH may pro- foundly influence the catalytic properties of LDH reactions (Winer& Schwert, 1958). Myctophid (H. atratum) white muscle LDH exhibited a decrease in Km with decreasing temperature (Fig. 1). Such an effect may act to stabilize the reaction by increasing the enzyme-substrate affinity to compensate for the decrease in thermal energy. Similar results have been noted for LDH as well as other enzymes (Hochachka & Lewis, 1971 ; Hochachka & Somero, 1968; Somero, 1969; Somero, 1973; Moon, 1972; Gerez De Burgos, 1973; Hochachka & Lewis, 1970). Stabilizing glycoly- tic rates in white muscle tissues by adjusting the Km of LDH for pyruvate to compensate for rapid en- vironmental changes in temperature and possibly in pH could be of great selective advantage to such fish as myctophids.

The pH influence on white muscle LDH in mycto- phid fish may be of adaptive significance as well. Values of Km were consistently highest when assayed at pH 8.0 (Fig. 1). Similar results have been reported for liver LDH from brook trout (Hochachka & Lewis, 1971) and from a snake (Gerez De Burgos, 1973). If the intracellular pH of H. atratum increased as these fish migrated to daytime depths, then the lowest tem- peratures these fish experience should produce the most alkaline pH. Although the Km of H. atratum white muscle LDH was highest at pH 8.0, this may be compensated for by behavior. Barbara (1970) has observed that myctophid fish are quite lethargic, actually not moving at all, when at daytime depths. An elevated Km for LDH may not be a disadvantage under such conditions. While migrating upward toward warmer water, the intracellular pH should de- crease as water temperature increases. The lowest Km values for H. atratum white muscle LDH were found at pH 7.0 (Fig. 1). Myctophid fish have been observed to be quite active in surface waters during night hours. Gut contents often reveal freshly ingested food. Low Km values for white muscle LDH could be ad- vantageous to fish which are relying heavily on these tissues to provide energy for active swimming.

Values o f Qt0 calculated for H. atratum white muscle LDH are near unity at substrate concen- trations which are considered physiological, and in- crease with increasing substrate concentration (Table 1). Physiological concentrations of pyruvate are believed to range from 0.01 to 0.1 mM (Somero & Hochachka, 1969; Freed, 1971; Mayerle & Butler, 1971). When pyruvate concentrations are low (at physiological levels), it is evident that LDH activity is only slightly affected by temperature, reaction vel- ocity being regulated by enzyme-substrate affinity. When substrate concentrations become saturating, the reaction velocity is regulated to a greater extent by thermodynamic effects, as is indicated by increased Qlo values. Gerez De Burgos et al. (1973) have reported similar effects with snake LDH, as have Hochachka & Somero (1968) with LDH from several fish; Hochachka & Lewis (1971) with trout liver LDH; Moon (1972) with pig heart isocitrate de- hydrogenase; and Hochachka & Lewis (1970) with trout liver citrate synthase.

Arrhenius plots at varying pyruvate concentrations support the Qlo data from Table 1. As pyruvate con- centration is reduced toward physiological levels,

Temperature and pH effects on catalytic properties 35

slopes of the Arrhenius plots decrease, indicating a de- crease in thermodynamic effects on reaction velocity (Fig. 4). At temperatures between 25 and 35°C and at physiological substrate concentrations the reaction velocity is nearly independent of temperatue.

Flying fish white muscle LDH

Results obtained with white muscle LDH from the flying fish Exocoetus volitans demonstrate that posi- tive thermal modulation may also take place in the white muscle tissues of these fish (Figs. 2 and'3). Fly ' ing fish are not diurnal migrators and hence do not experience the environmental fluctuations in tempera- ture that myctophid fish do. Apparently flying fish such as E. volitans have the capacity to compensate for decreasing thermal energy by increasing en.zyme- substrate affinity. Such ability in these fish is a curi- osity since they essentially inhabit a rather static en- vironment in regard to temperature. The unique anaerobic capability of flying fish white muscle tissue may be responsible for the observed direct variation of Km with temperature, although the necessity for such compensatory ability in these fish is not clear. Somero & Hochachka (1969) have found that enzyme-substrate affinity varies inversely with tem- perature over a large portion of a species' range of habitat temperatures for all poikilotherm enzymes examined. Such an ability may be characteristic of poikilotherms in general.

Surface temperatures in waters where flying fish specimens were. collected were near 27°C. Since these may be considered surface-dwelling fish and are not known to occur in deep water, their lower environ- mental thermal limit is not likely to be much below 27°C. At a depth of 450 m the temperature was 8°C. This temperature and depth is likely to be well below the thermal and spacial habitat of E. volitans. The decrease in Km with temperature between 15 and 5°C at pH 7.4 (Fig. 2) represents a condition unknown in most cases where the Km varies with temperature. Generally the Km will vary with-temperature in such a way that at temperatures near or beyond the limits of an organism's thermal habitat the Km will increase (Somero, 1969). This was not observed when assays were performed at 5°C with flying fish white muscle LDH.

Attempts to duplicate Km data from flying fish specimens 1 and 2 were not successful. Hochachka & Lewis (1971) have found that the enzyme concen- tration is itself an important determinant of the apparent Km. Increasing enzyme concentration leads to increasing Km values. Assays performed on the first flying fish specimen (Fig. 2) involved 2 g of white muscle tissue while those performed on the second specimen (Fig. 3) involved 1.22 g of white muscle tis- sue. The results of Hochachka & Lewis (1971) may in part explain the differences in Km observed for the two flying fish specimens. The abrupt increase in E-S affinity in Fig. 2 at pH 8.0 between 25 and 35°C would not be due to differences in enzyme concen- tration alone and should be considered tentative since efforts to duplicate the data were not successful.

The Qlo data for flying fish white muscle LDH is consistent with Qlo data from myctophid white muscle. Values were again found to be near 1.0 at physiological substrate concentrations indicating that

regulation of reaction velocity is dependent on enzyrne-substrate affinity under such conditions (Tables 2 and 3). Decreasing slopes of Arrhenius plots (Fig. 5) also indicate that reaction velocities become less affected by increasing temperature at physiologi- cal substrate concentrations in flying fish.

Acknowled#ements---I wish to thank Dr. George Pick- well for his support and advice through the course of this study performed at the Naval Undersea Center, San Diego, California, U.S.A.

Special thanks are extended to Mr. Frank Shipp for his aid in solving technical problems encountered during this study, and to Mr. William Friedl for his help in taxonomic identification of specimens.

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