Fish Physiology and Biochemistry vol. 3 no. 3 pp 151-162 (1987) Kugler Publications, Amsterdam/Berkeley
Distribution and properties of lactate dehydrogenase isoenzymes in red and white muscle of freshwater fish W. Wieser, R. Lackner, S. Hinterleitner and U. Platzer Institut fur Zoologie, Abteilung Zoophysiologie, Universitat Innsbruck, Technikerstrafle 25, A-6020 Innsbruck, Austria Keywords: salmonidae, coregonidae, cyprinidae, pyruvate reductase and lactate oxidase activity, temperature relationship of pyruvate and lactate affinity, starch gel electrophoresis
Abstract The distribution and kinetics of LDH isoenzymes in red and white muscles of 5 species of salmonids, 4 species of cyprinids and one coregonid species were studied. In all species the white muscles are characterized by the occurrence of only the most cathodic isoenzymes, or groups of isoenzymes. The red muscles contained either the full set of isoenzymes (cyprinids) or a selection in which the anodic forms dominated (salmonids, coregonid). The most striking difference between the two types of muscle was met in Coregonus sp. The temperature profiles of pyruvate affinity are similar in all species of fish studied. On the other hand, Km(pyr) values and degree of pyruvate inhibition are closely related and vary greatly with temperature, with the taxonomic position (and thus biology) of the species, and with electrophoretic mobility of the isoenzyme. Highest affinity and strongest inhibition occurred in the anodic (H 4 ) isoenzymes of cyprinids at low temperature; lowest affinity and zero inhibition in the cathodic isoenzymes (M,,4 - M/ 4) of salmonids and coregonids at high temperature. In salmonids the more recently duplicated loci of the M-group of isoenzymes possess identical Km values at all temperatures, whereas the two older M and H loci differ greatly in this respect. Thus the more recent duplication of LDH loci in salmonids and coregonids may be seen as a mechanism by which the tetramers required for LDH activity can be constructed from more closely related subunits than are provided by the older M and H loci. Some problems in connection with the determination of the kinetic constants of the lactate oxidase reaction are discussed and it is suggested that an alkaline, pyruvate trapping system provides conditions which are more realistic than those of other assay systems. The Km(lactate) values found are in the biological range and, at 20°C, provide further circumstantial evidence that the red muscles of fish should be capable of oxidizing the lactate produced by the white muscles during strenuous exercise. At 4°C the Km(lactate) values are abnormally high in all muscle preparations and thus are not correlated with the Km(pyruvate) values which are lowest at this temperature.
Introduction The functional separation of the isoenzymes of lactate dehydrogenase (LDH) has been the subject of discussion for about 25 years (Kaplan and Ciotti 1961). The most widely held theory states that the
M-type isoenzyme is especially geared to serve as a pyruvate reductase in tissues that are dependent on anaerobic glycolysis, whereas the properties of the H-type isoenzyme make this enzyme more efficient for the oxidation of lactate in tissues with an aerobic metabolism (Everse and Kaplan 1975). Al-
152 though this interpretation has been challenged (Vesell 1975) the kinetic differences between the H4 and M4 tetramers are clearcut: H 4 (= B4) possesses high affinity for pyruvate and is inhibited by high pyruvate concentrations, whereas M4 (= A4) shows low affinity for pyruvate and is not inhibited by high concentrations of this metabolite. If these differences in kinetic properties of the isoenzymes in vitro reflect their properties in vivo the distribution of isoforms may be used to characterize the metabolism of living tissues. This is a well-known approach that has been used, for example, in characterizing the energy metabolism of skeletal and heart muscle in mammals (Everse and Kaplan 1975). The distribution of the isoenzymes of LDH has also been studied extensively in the tissues of fish (Bouck and Ball 1968; Whitt et al. 1973; Wilson et al. 1975; Lim et al. 1975; Frankel 1980; Panepucci et al. 1984 and many others), but it seems to us that better use could be made of this molecular tool in defining the metabolic roles of different types of swimming muscles in these animals. There are two reasons for this supposition: Firstly, it has been argued (Braekkan 1956; Wittenberger and Diaciuc 1965; Wittenberger 1968) that differences in the properties of LDH might be due to the different ways in which lactate is handled by the two major types of muscle in fish, red fibres being able to oxidize directly the lactate produced by the fast glycolytic white fibres. Despite several more recent efforts (Bilinski and Jones 1972; Wittenberger et al. 1975; Hulbert and Moon 1978) a definite answer as to the metabolic capacity of red muscles in fish has not yet been given. Secondly, the relationship between swimming effort and energy metabolism differs in different species of fish. For example, in cyprinids lactate begins to accumulate in muscle tissue at relatively modest swimming speeds (Smit et al. 1972; Wieser et al. 1986) whereas in the rainbow trout lactate production commences in a burst-like fashion only at high swimming speeds (Brett 1972). Since it is more than likely that the distribution and the kinetic properties of the isoenzymes of LDH reflect the organization of metabolism in the swimming muscles a comparative investigation of these features might provide information on both
aspects of muscle physiology mentioned above. In the investigation to be reported we have used the patterns of distribution and of substrate affinities of the isoenzymes of LDH as a tool to define certain properties of energy metabolism in the swimming muscles of different species of freshwater fish. The choice of fish was dictated by the interests of our group in providing comparative data on the physiology and biochemistry of representatives of the two most important families of freshwater fish in European waters, Cyprinidae and Salmonidae (see, e.g. Hofer et al. 1975; Hinterleitner et al. 1986). Inclusion of a member of the Coregonidae was precipitated by the finding (Hinterleitner et al. 1986) that species of Coregonus are characterized by extremely high LDH activities in the white muscles which reflects their striking sprinting qualities and separates them from members of the other two families of teleosts. In the last paper quoted more comparative information on muscle and other enzymes in representatives of the three families of fish can be found.
Material and methods Fish Adults of the following species were examined: Salmonids: Brook charr, Salvelinus fontinalis; alpine charr, S. alpinus; lake trout, Salmo trutta lacustris; brook trout, S. trutta fario; rainbow trout, S. gairdneri.These species were bred in a fish farm near Innsbruck (Institut fur Fischforschung, Thaur), or were collected in various Tyrolean lakes and rivers. Cyprinids: Roach, Rutilus rutilus; bleak, Alburnus alburnus; chub, Leuciscus cephalus; rudd, Scardinius erythrophthalmus, from lakes near Innsbruck (Piburger See, Seefelder See) and from Mondsee, Upper Austria. Carp, Cyprinus carpio, were obtained from a local fish breeder. Preparationof tissues and extraction of enzymes The fish were either used immediately or maintained for several weeks in running water at approxi-
153 mately 12°C (all species except the carp) or at 20°C (C. carpio), and fed various types of dry food. No effects of the holding and feeding conditions of the fish on enzyme properties were ever detected, except the effect of starvation on Vmax, referred to in a previous paper (Hinterleitner et al. 1986). However, Vmax was not an object of study in the present investigation. The fish were killed by a blow on the head, skinned, and red and white muscles dissected. The red muscles were carefully removed along the lateral line of the fish and separated from adherent white fibres under a dissecting microscope. The piece of white muscle was always taken from the same body region, i.e. subdorsally, immediately behind the posterior edge of the dorsal fin. Weighed piece of muscle were frozen at -70°C. For enzyme extraction these pieces were homogenized in 20 vol. of ice cold phosphate buffer, 0.067 M, pH 7.5, with a Polytron homogenizer. The homogenates were centrifuged at 25000 g for 20 minutes, the supernatant separated into aliquots which were kept frozen at -70°C until use. After thawing the homogenate was applied to a Sephadex G-25 M (Pharmacia) column. The combined protein fraction was used for the enzyme assays.
Separation of isoenzymes Individual isoenzymes were separated by preparative isoelectric focussing. About 0.5 to 2.0 g of tissue were homogenized in distilled water and centrifuged. The supernatant was dialyzed against distilled water and mixed with the sucrose gradient of a LKB 110 ml Ampholine column containing ampholytes pH 5-8. Isoelectric focussing was performed at 4°C, with the anode at the bottom of the column, at a constant power of 3 W, maximum voltage set to 1000 V. After 24 hours focussing was complete and fractions of 1.5 ml were collected and assayed for LDH activity and pH. For horizontal starch gel electrophoresis 12% gels were used, containing 10% (w/v) sucrose and 6.77o (v/v) electrode (=bridge) buffer (0.13 M Tris, 0.043 M citrate, pH 7). Small pieces of filter paper were soaked with the samples and inserted in a cut slot of the gels. Gels were run at a constant
voltage of 10 V/cm for approximately 16 hours. The sample application papers were removed after the first hour of electrophoresis. The sliced gels were stained for LDH activity (Siciliano and Shaw 1976). Hydrolyzed starch was obtained from Electrostarch Comp., U.S.A., or from Sigma.
Enzyme assays LDH activity was assayed in both directions, pyruvate reducing and lactate oxidizing, at 4, 8, 12, 16, 20 and 24°C. Pyruvate reductase activity. Phosphate buffer 0.067 M, with 0.15 mM NADH and varying concentrations of sodium pyruvate. The pH was adjusted to 7.5 at 20°C and left to vary with experimental temperature. However, since the second dissociation constant of phosphate groups is unaffected by temperature the pH of phosphate buffers is also unaffected by temperature in the physiological range, i.e. dpH/dT = 0 (Wilson 1977). We checked this expectation (and found it fulfilled) by measuring the pH-value of the buffer over the range of experimental temperatures employed. L-lactate oxidase activity. Measured in this direction two problems arise. Since the equilibrium constant of the reaction pyruvate + NADH + H + = lactate + NAD + in phosphate buffer at pH 7.0 and 25°C is about 3 x 10 12 (Neilands 1952; Hakala et al. 1956) the reaction will proceed in the direction of lactate oxidation only when the concentration of NAD + and lactate are high. For the latter a value of at least 5 mM has been suggested (Everse and Kaplan 1975). When used for the determination of lactate it is customary to pull the reaction 'to the left' by trapping both H + and pyruvate with an alkaline buffer (usually glycine-NaOH, pH 9.0-10.0) containing hydrazine or semicarbazide (Bergmeyer 1974). Such a system is much less unphysiological
154 than it may appear at first, because a) neither hydrazine nor semicarbazide seem to have an effect on the initial velocity of the lactate oxidase reaction (Neilands 1952; Hakala et al. 1956; Winer and Schwert 1958); b) the pH optimum of the lactate oxidase reaction is around 9.0 (Winer and Schwert 1958); c) as in the living cell the accumulation of pyruvate is prevented. The last point is of particular relevance when the LDH isoforms studied are sensitive to pyruvate inhibition, as is the case with many isoenzymes of poikilotherms at low temperature. Pyruvate trapping systems have been used for establishing the kinetic constants of the lactate oxidase reaction (Hakala et al. 1956; Winer and Schwert 1958). More recent authors, on the other hand, appear to have been worried about what they considered the unphysiological nature of these assay conditions and have used more neutral buffers, usually Tris/HCI pH 7.4, without pyruvate trapping reagents (Hulbert and Moon 1978; Driedzic et al. 1985). In our hands these conditions led to rapid inhibition of some isoforms of fish LDH when the rate of lactate oxidation was measured. In consequence, and in view of what has been said above, we employed the following assay system for measuring the kinetic constants of the lactate oxidase reaction: Buffer: Dissolve 0.94 g glycine in 20 ml distilled water; set pH to 9.0 by adding 1.5-2.0 ml of a 24°7o solution of hydraziniumhydroxide (Merck); make up volume to 25 ml which gives a buffer concentration of 0.5 M. Assay: Buffer, NAD + to give an end concentration of 4.8 mM, varying concentrations of L-lactate (lithium salt), enzyme solution. The pH of the buffer was set at 20°C and left to vary with temperature, increasing to 9.11 at 4°C, decreasing to 8.93 at 24°C. This results in a dpH/dT(°C) value of 0.007 which is much lower than the 0.015-0.020 characteristic of imidazol buffers and the body fluids of ectothermic animals (Wilson 1977, Reeves 1977). Thus it may be assumed that under the conditions chosen both the reducing and the oxidizing reaction catalyzed by LDH proceeded at a more or less constant pH value, 7.5 and 9.0 respectively, over the range of experimental temperatures em-
Fig. . Starch gel electrophoresis od LDH isoenzymes from homogenates of red (R) and white (W) muscle of representatives of three families of freshwater fish, a coregonid (Coregonus sp.), a salmonid (Salvelinusfontinalis), and two cyprinids (Rutilus rutilus and Cyprinus carpio). S = start.
ployed. The kinetic constants were calculated by means of a programme based on the graphic method of Eisenthal and Cornish-Bowden (1974).
Results Isoenzyme patterns in red and white muscle from different species (Fig. I) Following the nomenclature suggested by Bailey et al. (1976) we shall designate the two major loci of LDH in fish muscle by LDH M and LDH H (A and B according to a different nomenclature), the basic set of five isoenzymes thus ranging from M4 to H4 . The recent duplication of loci observed in salmonids (and - as shown below - in coregonids) are designated Ma, Ms , H,, H, and the various alleles for loci which show polymorphism by capitalized Arabic superscripts, e.g. LDH H4Aand LDH H4B. In all species investigated the white muscles are characterized by the occurrence of only the most cathodic isoenzymes or group of isoenzymes. In cyprinids only M4 and , sometimes but always much more weakly expressed, M3HI, in salmonids and coregonids only the cathodic group Ma 4 to M4, are present. The red muscles contained either the
155
full set of isoenzymes (cyprinids) or a selection in which the anodic isoenzymes dominated. The most striking difference between the two types of muscles was met in Coregonus sp. in which the occurrence of the M-group and the H-group of isoenzymes was mutually exclusive. In salmonids the H-set was dominant in the red muscles but there were also members of the M-set and intermediate bands of the Ma Ha,, type present. This is in accordance with the findings of Bouck and Ball (1968). In C. carpio red muscle the H4 isoenzyme (in our population represented by two alleles, HA and HB) was always much more strongly expressed than the other isoenzymes, whereas in the red muscles of R. rutilus, L. cephalus, A. alburnusand S. erythrophthalmus the five isoenzymes occurred with more or less equal activity. In one population of R. rutilus (Seefeld) the H locus was represented by two alleles.
Table 1. Pyruvate affinities (Km values in mM) at 24°C of LDH in homogenates of red and white muscles, or of separated individual isoenzymes, in different species of fish. Km(pyr) (mM) at 24°C white muscles or M4
red muscles or H4
Rutilus rutilus (separated isoenzymes) (homogenate)
0.35 0.38 + 0.13
0.1 0.26 ± 0.06
Cyprinus carpio (homogenate)
0.48 + 0.08
0.1
Salvelinus fontinalis (homogenate) Coregonus sp. (homogenate)
0.14
0.95 0.89 ± 0.23
+ 0.03
0.3
+ 0.11
Either mean values + S.D. of three experiments, or the means of two experiments, are given.
Kinetic properties of lactate dehydrogenases Temperature profiles of pyruvate affinity. The cathodic isoenzyme(s) which dominate the white muscles of all species investigated displayed very similar temperature profiles of pyruvate affinity (Figs. 2-4). An increase in Km(pyr) from 4 to 24°C was observed in all species studied, although these differ widely in ecology and temperature preference. In salmonids the a and 3 loci of the M-group possess identical temperature relationships (Fig. 3), thus supporting the notion (Bailey et al. 1976) that this group arose through fairly recent duplication of the original M locus. The temperature profile of the most anodic isoenzyme (H4 ) or group of isoenzymes (h. 4 to H4), present only in red muscles, is also identical in all species of fish investigated. The temperature dependence of pyruvate affinity of this isoenzyme is generally weaker than that of the M isoenzyme but its major distinctive feature is its much lower Km value, the difference being most clearly expressed at the highest experimental temperatures. This is illustrated by comparing the two separated isoenzymes, or sets of isoenzymes, or the homogenates of red and white muscles from those species in
which each type of muscle is dominated by one type, or set, of isoenzyme(s) (Table 1). The latter condition holds for some salmonids and for Coregonus sp., but also for C. carpio in which species H4 was the dominant isoenzyme of the red muscle, whereas in the other four species of cyprinids all five isoenzymes occurred with about equal activity in this tissue (Fig. 1). In accordance with this distribution the temperature profile of pyruvate affinity is very similar in red muscle homogenates of the four temperate water cyprinids (Fig. 4), whereas it is characteristically different in the warm water species, C. carpio (Fig. 2). The same difference holds for the red muscle of the salmonid species investigated which display great variability both in isoenzyme distribution and in the temperature relationship of Km-values. Two extreme cases of the latter have been illustrated in Fig. 4. It is concluded that the temperature profile of pyruvate affinity in the red muscle of fish is mainly determined by the extent to which the cathodic isoenzyme(s) contribute(s) to total LDH activity. Substrate inhibition. By comparing the activities of LDH at high and low pyruvate concentrations one
156
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4 8 12 16 20 24 4 8 12 16 20 24 ?mperature (C) Fig. 2. Temperature profiles of Kn, values (mM pyruvate) of the lactate dehydrogenases from red and white muscle homogenates, or of isolated M4 and H4 isoenzymes, of cyprinids. Mean values and standard deviations of several samples as indicated. To the right the distribution and relative activity of isoenzymes after starch gel electrophoresis is illustrated for red and white muscle of roach (upper panel), carp (lower panel) and for the M4 and H4 isoenzymes of roach (middle panel).
obtains a measure of the strength of substrate inhibition of this enzyme. When a quotient R is defined as the ratio of activities at 3.0 and 0.1 mM pyruvate concentrations, Fig. 4 attests to the very close rela-
tionship between Km and R values in all species and tissues examined. The higher pyruvate affinity the higher also the degree of substrate inhibition, i.e. the lower R. Thus highest affinity and strongest
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temperature (°C) Fig. 3. Temperature profiles of K,1, values (mM pyruvate) of the lactate dehydrogenases from red and white muscle homogenates of a salmonid (upper panel) and a coregonid (lower panel). In the salmonid, Salvelinusfontinalis, the temperature profiles of two isoenzymes of the M-group are also shown; h = homogenate. The distribution and relative activity of the LDH isoenzymes in the two types of muscle are indicated by the schematized electropherograms to the right.
158
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temperature (C) Fig. 4. Temperature profiles of Km, (mM pyruvate) and R values (ratio of enzyme activities at 3.0 and 0.1 mM pyruvate), in three families of freshwater fish, comprising data from 4 species of cyprinids, 5 species of salmonids, and one species of coregonid. Mean values and standard deviations are given. The salmonid data are summarized for white muscle homogenates only since the greater interspecific variability of isoenzyme composition of red muscle also implies greater variability of the temperature relationship of pyruvate affinity in this tissue (see text). The inset illustrates these differences by presenting the temperature profiles of pyruvate affinity in red muscle homogenates of Salmo gairdneriand Salmo truttafario.
159 Table 2. Ki values (mM lactate) of lactate oxidase reaction in red and white muscles of three species of freshwater fish measured at 2 temperatures. red muscle
Salvelinusfoninalis Coregonus sp. Rutilus rulilus
white muscle
4°C
20°C
4oC
20°C
11.0 + 1.3 4.9 + 0.8 6.0 + 1.9
7.5 + 0.8 3.4 + 0.5 2.5 + 0.5
14.7 + 0.8 12.3 + 0.9 14.8 + 1.4
10.7 1.0 8.5 + 1.7 3.7 + 0.9
Assay medium glycine-NaOH with hydrazine, pH 9.11 (4°C)-9.0 (20°C). Means and standard deviations of three independent samples.
inhibition is displayed by the cyprinid isoenzymes at low temperature, lowest affinity and zero inhibition by the salmonid and coregonid isoenzymes at high temperature. Lactate affinity. We measured the kinetics of the lactate oxidase reaction under alkaline conditions with pyruvate trapping at 4° and 20°C, in the red and white muscles of R. rutilus, S. fontinalis, and Coregonus sp. (Table 2). At 200 C the distribution of the Km-values for L-lactate corresponds to what was to be expected on the basis of the K,,-values for pyruvate; that is, substrate affinity is always higher in red than in white muscle fibres, the degree of difference reflecting the distribution of the isoenzymes in the muscle tissues of the three families investigated (Fig. 1). For example, the greatest difference in lactate affinity between red and white muscles occurs in Coregonus sp., the species with the greatest difference in the distribution of LDH isoenzymes between these tissues (Fig. 1). However, the results of the kinetic measurements at 4°C were totally unexpected and are at variance with the few data on the temperature relationship of the lactate oxidase reaction which we were able to locate in the literature (Henry and Ferguson 1985). Whereas in all species and tissues pyruvate reduction is characterized by a direct relationship between Km-value and temperature, the opposite is true for lactate oxidation - at least between 4° and
20°C (Table 2). Under all assay conditions tested (see Methods) the Km-value for lactate oxidation proved to be higher at 4 than at 20°C.
Discussion The distribution of LDH isoenzymes in red and white muscle fibres conforms to a well-known pattern (see Bouck and Ball 1968), the fast glycolytic fibres being characterized by the dominance of the most cathodic isoenzyme - or group of isoenzymes - whereas in the oxidative fibres a more varied pattern, always including the most anodic isoenzymes, prevails (Fig. 1). No attention seems to have been paid in the literature to the more subtle variations of this general pattern in different taxa of fish, and whether these varations might not enable us to draw biologically or physiologically meaningful conclusions. For example, there could be significance in the fact that in predatory species (coregonid, salmonids) the red muscle fibres are very clearly dominated by the anodic group of isoenzymes. In fact, in Coregonus sp., the most pronounced 'sprinter' of all species investigated (Hinterleitner et al. 1986), no overlap is observed between the isoenzymes of red and white muscle fibres, the former being characterized by an anodic, the latter by a cathodic, set of isoenzymes. In 'stayers', like the more slowly swimming cyprinids, the red muscle fibres are characterized by a more complete set of isoenzymes, and more moderate sprinters, like the salmonids, occupy an intermediate position in this respect. Even within one family difference can be found. For example, in the carp, the isoenzyme patterns of red and white muscles are more clearly distinguished than in the four other cyprinids investigated (Fig. 1). On the strength of other findings we believe that a functional explanation for this difference will be found, perhaps connected with the
160 different temperature relationships of the species concerned. The most complete separation of isoenzymes between red and white muscles is due to the duplication of individual LDH loci which allows either of the original M and H loci to express a set of five isoenzymes (Lim et al. 1975; Bailey et al. 1976; Klar et al. 1979; DiMichele and Powers 1982; Panepucci et al. 1984). One could visualize this more recent process of duplication as a mechanism by which the tetramers required for LDH activity can be constructed from closely related subunits. For example, within the M group of S. fontinalis the a and 3 loci exhibit identical temperature profiles for pyruvate affinity, whereas the profiles of the old M and H loci differ greatly from each other (Fig. 3). Thus, if for functional reasons a homogeneous population of tetramers were required the M, MO series of isoenzymes would be more suitable than the M,H series. This is so because the more closely related series of isoenzymes would be able to carry out a specialized task without the additional intervention of posttranslational control (Whitt 1970; Rosenberg 1971; Vesell 1975). This may be the reason why the more recent duplications of the M and H loci occur preferentially in fast-swimming carnivorous fish in which the sustained and the burst-like mode of swimming activity are more clearly separated than in herbivores and omnivores. However, despite their more homogeneous substrate relationship new and subtle differences have already appeared between the isoenzymes of the more recently duplicated alleles of LDH (Lim et al. 1975; Kao and Farley 1978; Klar et al. 1979). In fact, a slight shift in expression of white muscle LDH isoenzymes in the direction of the more aerobic B' subunit (corresponding to our 3 subunit) was observed when rainbow trout were subjected to sustained swimming activity for 200 days (Davie et al. 1986). In order to characterize the kinetic differences between the LDH's of red and white muscle fibres we used experimental temperature as an analytical tool (Figs. 2-4). We did not intend a detailed study of the temperature relationships of substrate affinities since for this purpose the temperature regimes of the environments and of the holding tanks were
not sufficiently well known or controlled. However, it should be pointed out that the up to threefold difference in pyruvate affinity between cyprinids on the one hand, salmonids and coregonids on the other hand (Fig. 4), is difficult to interpret in terms of the 'stable Km hypothesis' advanced by Yancey and Somero (1978). These authors held that in fish from a wide variety of habitats the K(pyr) values are regulated between 0.2 and 0.3 mM at the approximate range of temperature of the species. This conclusion is at variance with the large difference in Km-values between sympatric species (for example R. rutius, Coregonus sp., S. gairdneri which co-occur in many Austrian lakes). On the other hand, two cyprinids with very different temperature relationships, C. carpio and R. rutilus, display the same temperature profile of pyruvate affinity (Fig. 2). Rather it appears that differences in the substrate affinity of LDH reflect differences in the metabolism of swimming muscles. This could account for the kinetic differences not only between red and white fibres but also between the LDH's of the three families of fish studied. For example, in cyprinids lactate production is turned on at lower fractions of maximum swimming speeds than in salmonids (Driedzic and Kiceniuk 1976; Jones 1982; Wieser et al. 1986). Thus the LDH of cyprinid muscles should act as a lactate oxidase at lower concentrations of lactate than the LDH of salmonid muscles. This is borne out by the Km values of the lactate oxidase reaction in red muscles which are about three times higher in S. fontinalis than in R. rutilus (Table 2). One would have expected the coregonid species to resemble the salmonid in this respect, but its Km value for lactate oxidation is only slightly higher than that of R. rutilus red muscle. On the other hand, the white muscles of Coregonus sp. behave, as far as their affinity for lactate is concerned, like those of a salmonid species. There may be a biological message hidden in these differences. The Km values of lactate oxidation, as determined by our assay method, are in the range to be expected from the concentrations of lactate in fish blood and muscle after strenuous exercise. Whenever sampling artifacts are avoided by applying the correct procedure of freeze clamping (Wieser et al.
161 1986) concentrations of not more than 20 ptmol lactate per gram fresh weight of muscle, or ml of blood, are found both in salmonids (Black 1957; Driedzic and Kiceniuk 1976) and in cyprinids (Driedzic and Hochachka 1976; Wieser et al. 1986). This agrees well with Km values of from 2.5 + 0.5 to 7.3 + 0.9 mM lactate for the red muscle LDH's of our species measured at 20°C (Table 2). Km values exceeding 20 mM lactate, as reported, e.g., by Driedzic et al. (1985) for the LDH's of fish hearts would appear to be artifacts due to the assay method used by these authors. We have no explanation for the temperature anomaly of lactate affinity of the LDH's in our preparations as summarized in Table 2. If this finding reflects the conditions in vivo then the temperature relationship of pyruvate reduction must be different from that of lactate oxidation, with the consequence that the rate of lactate production during strenuous exercise may be unaffected by low temperature, whereas recovery should be slowed down by this factor. The former supposition is almost certainly correct (Brett 1964; Wieser et al. 1985), for the latter we have no evidence. At any rate, the difference in temperature relationship between the pyruvate reducing and the lactate oxidizing reaction cannot be due to differences in the pH/T relationship of the two assay media employed since the dpH/dT value was practically zero for both reactions (see Materials and methods). In conclusion it can be said that the properties of LDH in the red muscles of teleosts strongly indicate that this tissue plays an important role in the oxidation of lactate produced during locomotory activity in the white muscles, and that the extent of this ability depends on the biology of the species in question, as well as on temperature and other environmental factors.
Acknowledgement This research was supported by the 'Fonds zur Forderung der wissenschaftlichen Forschung in Osterreich', project no. S-35/04.
References cited Bailey, G.S., Tsuyuki, H. and Wilson, A.C. 1976. The number of genes for lactate dehydrogenase in salmonid fishes. J. Fish. Res. Board Can. 33: 760-767. Bergmeyer, H.U. 1974. Methoden der Enzymatischen Analyse. 3.Aufl. Verlag Chemie, Weinheim/Bergstrasse. Bilinski, E. and Jones, R.E.E. 1972. Oxidation of lactate to carbon dioxide by rainbow trout (Salmo gairdneri). J. Fish. Res. Board Can. 29: 1467-1471. Black, E.C. 1957. Alterations in the blood level of lactic acid in certain salmonid fishes following muscular activity. l.Kamloops trout (Salmo gairdneri). J. Fish. Res. Board Can. 14: 117-134. Bouck, G.R. and Ball, R.C. 1968. Comparative electrophoretic patterns of lactate dehydrogenase in three species of trout. J. Fish. Res. Board Can. 25: 1323-1331. Braekkan, O.R. 1956. Function of red muscle in fish. Nature, Lond. 178: 747-748. Brett, J.R. 1964. The respiratory metabolism and swimming performance of young sockeye salmon. J. Fish. Res. Board Can. 21: 1183-1226. Brett, J.R. 1972. The metabolic demand for oxygen in fish, particularly salmonids, and a comparison with other vertebrates. Resp. Physiol. 14: 151 170. Cahn, R., Kaplan, N., Levine, L. and Zwilling, E. 1962. Nature and development of lactate dehydrogenase. Science 136: 962 969. Davie, P.S., Wells, R.M.G. and Tetens, V. 1986. Effects of sustained swimming on rainbow trout muscle structure, blood oxygen transport, and lactate dehydrogenase isoenzymes. Evidence for increased aerobic capacity of white muscle. J. Exp. Zool. 237: 159-171. DiMichele, L. and Powers, D. 1982. Physiological basis for swimming endurance differences between LDH-B genotypes of Fundulus heteroclitus. Science 216: 1014-1016. Driedzic, W.R. and Hochachka, P.W. 1976. Control of energy metabolism in carp white muscle. Am. J. Physiol. 230: 579-582. Driedzic, W.R. and Kiceniuk, J.W. 1976. Blood lactate levels in free swimming trout (Salmo gairdneri) before and after exercise resulting in fatigue. J. Fish. Res. Board Can. 33: 173-176. Driedzic, W.R., Stewart, J.M. and McNairn, G. 1985. Control of lactate oxidation in fish hearts by lactate oxidase activity. Can. J. Zool. 63: 484 487. Eisenthal, R. and Cornish-Bowden, A. 1974. The direct linear plot. A new graphical procedure for estimating enzyme kinetic parameters. Biochem. J. 139: 715-720. Everse, J. and Kaplan, N.O. 1975. Mechanisms of action and biological functions of various dehydrogenase isoenzymes. In Isozymes, Vol. II, pp. 29-43. Edited by C.L.Markert, Academic Press, New York. Forstner, H., Hinterleitner, S., Mahr, K. and Wieser, W. 1983. Towards a better definition of "metamorphosis" in Coregonus sp.: Biochemical, histological and physiological data.
162 Can. . ish. Aquat. Sci. 40: 1224-1232. Frankel, J.S. 1980. Lactate dehydrogenase isoenzymes of the leopard danio, Brachidanio nigrofasciatus: their characterization and ontogeny. Comp. Biochem. Physiol. 67B: 133-137. Hakala, M.T., Glaid, A.J. and Schwert, G.W. 1956. Lactic dehydrogenase. II.Variation of kinetic and equilibrium constants with temperature. J. Biol. Chem. 221: 191-209. Henry, T. and Ferguson, A. 1986. Kinetic studies on the lactate dehydrogenase (LDH-5) isozymes of brown trout, Salmo trutta L. Comp. Biochem. Physiol. 82B: 95-98. Hinterleitner, S., Platzer, U. and Wieser, W. 1986. Development of the activities of oxidative, glycolytic and muscle enzymes during early larval life in three families of freshwater fish. J. Fish Biol., (in press). Hofer, R., Ladurner, H., Gattringer, A. and Wieser, W. 1975. Relationship between the temperature preferenda of fishes, amphibians and reptiles and the substrate affinities of their trypsins. J. Comp. Physiol. 99: 345-355. Hulbert, W.C. and Moon, T.W. 1978. The potential for lactate utilization by red and white muscle of the eel, Anguilla rostrata. L. Can. J. Zool. 56: 128-135. Jones, D.R. 1982. Anaerobic exercise in teleost fish. Can. J. Zool. 60: 1131-1134. Kao, Y.-H., Farley, J. and T.M. 1978. Purification and proper2 ties of allelic lactate dehydrogenase isozymes at the B locus in rainbow trout, Sah7oo gairdneri. Comp. Biochem. Physiol. 61B: 507-512. Kaplan, N.O. and Ciotti, M.M. 1961. Evolution and differentiation of dehydrogenases. Ann. N. Y. Acad. Sci. 94: 701-722. Klar, G.T., Stalnaker, C.B. and Farley, T.M. 1979. Comparative physical and physiological performance of rainbow trout, 2 Salllo gairdneri, of distinct lactate dehydrogenase B phenotypes. Comp. Biochem. Physiol. 63A: 229 235. Lim, S.T., Kay, R.M. and Bailey, G.S. 1975. Lactate dehydrogenase isozymes of salmonid fish: Evidence for unique and rapid functional divergence of duplicated H4 lactate dehydrogenases. J. Biol. Chem. 250: 1790 1800. Neilands, J.B. 1952. Studies on lactic dehydrogenase of heart. I.Purity, kinetics, and equilibria. J. Biol. Chem. 199: 373 381. Panepucci, L.L.L. de, Schwantes, M.L.B. and Schwantes, A.R. 1984. Loci that encode the lactate dehydrogenase in 23 species of fish belonging to the orders Cypriniformes, Siluriformes and Perciformes: adaptive features. Comp. Biochem. Physiol. 77B: 867-876. Reeves, R.B. 1977. The interaction of body temperature and acid-base balance in ectothermic vertebrates. Ann. Rev. Physiol. 39: 559-586. Rosenberg, M. 1971. Epigenetic control of lactate dehydrogenase subunit assembly. Nature New Biology 230: 12-14. Siciliano, M.J. and Shaw, C.R. 1976. Separation and visualisati-
on of enzymes on gels. In Chromatographic and Electrophoretic Techniques, Vol. 2, 4th ed., pp. 185-209. Edited by I. Smith. W. Heinemann, London. Smit, H., Amelink-Koutstaal, I.M., Vijvetberg, J. and von Vaupel-Klein, J.C. 1972. Oxygen consumption and efficiency of swimming in goldfish. Comp. Biochem. Physiol. 39A: 1-28. Vesell, E.S. 1975. Medical uses of isozymes. In Isozymes I1. Physiological Function, pp. 1-28. Edited by C.L. Markert, Academic Press, New York. Whitt, G.S. 1970. Developmental genetics of the lactate dehydrogenase isozymes of fish. J. Exp. Zool. 175: 1-36. Whitt, G.S., Miller, E.T. and Shaklee, J.B. 1973. Developmental and biochemical genetics of lactate dehydrogenase isozymes in fishes. In Genetics and Mutagenesis of Fish, pp. 243-276. Edited by J.H. Schroder, Springer Verlag Berlin, Heidelberg, New York. Wieser, W., Platzer, U. and Hinterleitner, S. 1985. Anaerobic and aerobic energy production of young rainbow trout (Sal,no gairdneri) during and after bursts of activity. J. Comp. Physiol. 155B: 485-492. Wieser, W., Koch, F., Drexel, E. and Platzer, U. 1986. "Stress" reactions in teleosts: effects of temperature and activity on anaerobic energy production in roach (Rutilus rutilus L.). Comp. Biochem. Physiol. 83A: 41-45. Wilson, T.L. 1977. Theoretical analysis of the effects of two pH regulation patterns on the temperature sensitivities of biological systems in nonhomeothermic animals. Arch. Biochem. Biophys. 182: 409 419. Wilson, F.R., Champion, M.J., Whitt, G.S. and Prosser, C.L. 1975. Isozyme patterns in tissues of temperature-acclimated fish. In Isozymes, Vol. 2, pp. 193-206. Edited by C.L. Markert. Academic Press, New York. Winer, A.D. and Schwert, G.W. 1958. Lactic dehydrogenase: the influence of pH on kinetics of the reaction. J. Biol. Chem. 231: 1065-1083. Wittenberger, G. 1968. Biologic du chinchard de la Mer Noire (Trachurus ,nediterraneus ponticus). XV. Recherches sur le metabolisme d'effort chex Trachurus et Gobius. Mar. Biol. 2:1 4. Wittenberger, G. and Diaciuc, I.U. 1965. Effort metabolism of lateral muscle in carp. J. Fish. Res. Board Can. 22: 13971406. Wittenberger, G., Coprean, D. and Morar, L. 1975. Studies in the carbohydrate metabolism of the lateral muscles in carp. (Influence of phloridizin, insulin and adrenaline). J. Comp. Physiol. 101: 161-172. Yancey, P.H.M. and Somero, G.N. 1978. Temperature dependence of intracellular pH. Its role in the conservation of pyruvate apparent K values of vertebrate lactate dehydrogenases. J. Comp. Physiol. 125: 135-141.
Distribution and properties of lactate dehydrogenase isoenzymes in red and white muscle of freshwater fish W. Wieser, R. Lackner, S. Hinterleitner and U. Platzer Institut fur Zoologie, Abteilung Zoophysiologie, Universitat Innsbruck, Technikerstrafle 25, A-6020 Innsbruck, Austria Keywords: salmonidae, coregonidae, cyprinidae, pyruvate reductase and lactate oxidase activity, temperature relationship of pyruvate and lactate affinity, starch gel electrophoresis
Abstract The distribution and kinetics of LDH isoenzymes in red and white muscles of 5 species of salmonids, 4 species of cyprinids and one coregonid species were studied. In all species the white muscles are characterized by the occurrence of only the most cathodic isoenzymes, or groups of isoenzymes. The red muscles contained either the full set of isoenzymes (cyprinids) or a selection in which the anodic forms dominated (salmonids, coregonid). The most striking difference between the two types of muscle was met in Coregonus sp. The temperature profiles of pyruvate affinity are similar in all species of fish studied. On the other hand, Km(pyr) values and degree of pyruvate inhibition are closely related and vary greatly with temperature, with the taxonomic position (and thus biology) of the species, and with electrophoretic mobility of the isoenzyme. Highest affinity and strongest inhibition occurred in the anodic (H 4 ) isoenzymes of cyprinids at low temperature; lowest affinity and zero inhibition in the cathodic isoenzymes (M,,4 - M/ 4) of salmonids and coregonids at high temperature. In salmonids the more recently duplicated loci of the M-group of isoenzymes possess identical Km values at all temperatures, whereas the two older M and H loci differ greatly in this respect. Thus the more recent duplication of LDH loci in salmonids and coregonids may be seen as a mechanism by which the tetramers required for LDH activity can be constructed from more closely related subunits than are provided by the older M and H loci. Some problems in connection with the determination of the kinetic constants of the lactate oxidase reaction are discussed and it is suggested that an alkaline, pyruvate trapping system provides conditions which are more realistic than those of other assay systems. The Km(lactate) values found are in the biological range and, at 20°C, provide further circumstantial evidence that the red muscles of fish should be capable of oxidizing the lactate produced by the white muscles during strenuous exercise. At 4°C the Km(lactate) values are abnormally high in all muscle preparations and thus are not correlated with the Km(pyruvate) values which are lowest at this temperature.
Introduction The functional separation of the isoenzymes of lactate dehydrogenase (LDH) has been the subject of discussion for about 25 years (Kaplan and Ciotti 1961). The most widely held theory states that the
M-type isoenzyme is especially geared to serve as a pyruvate reductase in tissues that are dependent on anaerobic glycolysis, whereas the properties of the H-type isoenzyme make this enzyme more efficient for the oxidation of lactate in tissues with an aerobic metabolism (Everse and Kaplan 1975). Al-
152 though this interpretation has been challenged (Vesell 1975) the kinetic differences between the H4 and M4 tetramers are clearcut: H 4 (= B4) possesses high affinity for pyruvate and is inhibited by high pyruvate concentrations, whereas M4 (= A4) shows low affinity for pyruvate and is not inhibited by high concentrations of this metabolite. If these differences in kinetic properties of the isoenzymes in vitro reflect their properties in vivo the distribution of isoforms may be used to characterize the metabolism of living tissues. This is a well-known approach that has been used, for example, in characterizing the energy metabolism of skeletal and heart muscle in mammals (Everse and Kaplan 1975). The distribution of the isoenzymes of LDH has also been studied extensively in the tissues of fish (Bouck and Ball 1968; Whitt et al. 1973; Wilson et al. 1975; Lim et al. 1975; Frankel 1980; Panepucci et al. 1984 and many others), but it seems to us that better use could be made of this molecular tool in defining the metabolic roles of different types of swimming muscles in these animals. There are two reasons for this supposition: Firstly, it has been argued (Braekkan 1956; Wittenberger and Diaciuc 1965; Wittenberger 1968) that differences in the properties of LDH might be due to the different ways in which lactate is handled by the two major types of muscle in fish, red fibres being able to oxidize directly the lactate produced by the fast glycolytic white fibres. Despite several more recent efforts (Bilinski and Jones 1972; Wittenberger et al. 1975; Hulbert and Moon 1978) a definite answer as to the metabolic capacity of red muscles in fish has not yet been given. Secondly, the relationship between swimming effort and energy metabolism differs in different species of fish. For example, in cyprinids lactate begins to accumulate in muscle tissue at relatively modest swimming speeds (Smit et al. 1972; Wieser et al. 1986) whereas in the rainbow trout lactate production commences in a burst-like fashion only at high swimming speeds (Brett 1972). Since it is more than likely that the distribution and the kinetic properties of the isoenzymes of LDH reflect the organization of metabolism in the swimming muscles a comparative investigation of these features might provide information on both
aspects of muscle physiology mentioned above. In the investigation to be reported we have used the patterns of distribution and of substrate affinities of the isoenzymes of LDH as a tool to define certain properties of energy metabolism in the swimming muscles of different species of freshwater fish. The choice of fish was dictated by the interests of our group in providing comparative data on the physiology and biochemistry of representatives of the two most important families of freshwater fish in European waters, Cyprinidae and Salmonidae (see, e.g. Hofer et al. 1975; Hinterleitner et al. 1986). Inclusion of a member of the Coregonidae was precipitated by the finding (Hinterleitner et al. 1986) that species of Coregonus are characterized by extremely high LDH activities in the white muscles which reflects their striking sprinting qualities and separates them from members of the other two families of teleosts. In the last paper quoted more comparative information on muscle and other enzymes in representatives of the three families of fish can be found.
Material and methods Fish Adults of the following species were examined: Salmonids: Brook charr, Salvelinus fontinalis; alpine charr, S. alpinus; lake trout, Salmo trutta lacustris; brook trout, S. trutta fario; rainbow trout, S. gairdneri.These species were bred in a fish farm near Innsbruck (Institut fur Fischforschung, Thaur), or were collected in various Tyrolean lakes and rivers. Cyprinids: Roach, Rutilus rutilus; bleak, Alburnus alburnus; chub, Leuciscus cephalus; rudd, Scardinius erythrophthalmus, from lakes near Innsbruck (Piburger See, Seefelder See) and from Mondsee, Upper Austria. Carp, Cyprinus carpio, were obtained from a local fish breeder. Preparationof tissues and extraction of enzymes The fish were either used immediately or maintained for several weeks in running water at approxi-
153 mately 12°C (all species except the carp) or at 20°C (C. carpio), and fed various types of dry food. No effects of the holding and feeding conditions of the fish on enzyme properties were ever detected, except the effect of starvation on Vmax, referred to in a previous paper (Hinterleitner et al. 1986). However, Vmax was not an object of study in the present investigation. The fish were killed by a blow on the head, skinned, and red and white muscles dissected. The red muscles were carefully removed along the lateral line of the fish and separated from adherent white fibres under a dissecting microscope. The piece of white muscle was always taken from the same body region, i.e. subdorsally, immediately behind the posterior edge of the dorsal fin. Weighed piece of muscle were frozen at -70°C. For enzyme extraction these pieces were homogenized in 20 vol. of ice cold phosphate buffer, 0.067 M, pH 7.5, with a Polytron homogenizer. The homogenates were centrifuged at 25000 g for 20 minutes, the supernatant separated into aliquots which were kept frozen at -70°C until use. After thawing the homogenate was applied to a Sephadex G-25 M (Pharmacia) column. The combined protein fraction was used for the enzyme assays.
Separation of isoenzymes Individual isoenzymes were separated by preparative isoelectric focussing. About 0.5 to 2.0 g of tissue were homogenized in distilled water and centrifuged. The supernatant was dialyzed against distilled water and mixed with the sucrose gradient of a LKB 110 ml Ampholine column containing ampholytes pH 5-8. Isoelectric focussing was performed at 4°C, with the anode at the bottom of the column, at a constant power of 3 W, maximum voltage set to 1000 V. After 24 hours focussing was complete and fractions of 1.5 ml were collected and assayed for LDH activity and pH. For horizontal starch gel electrophoresis 12% gels were used, containing 10% (w/v) sucrose and 6.77o (v/v) electrode (=bridge) buffer (0.13 M Tris, 0.043 M citrate, pH 7). Small pieces of filter paper were soaked with the samples and inserted in a cut slot of the gels. Gels were run at a constant
voltage of 10 V/cm for approximately 16 hours. The sample application papers were removed after the first hour of electrophoresis. The sliced gels were stained for LDH activity (Siciliano and Shaw 1976). Hydrolyzed starch was obtained from Electrostarch Comp., U.S.A., or from Sigma.
Enzyme assays LDH activity was assayed in both directions, pyruvate reducing and lactate oxidizing, at 4, 8, 12, 16, 20 and 24°C. Pyruvate reductase activity. Phosphate buffer 0.067 M, with 0.15 mM NADH and varying concentrations of sodium pyruvate. The pH was adjusted to 7.5 at 20°C and left to vary with experimental temperature. However, since the second dissociation constant of phosphate groups is unaffected by temperature the pH of phosphate buffers is also unaffected by temperature in the physiological range, i.e. dpH/dT = 0 (Wilson 1977). We checked this expectation (and found it fulfilled) by measuring the pH-value of the buffer over the range of experimental temperatures employed. L-lactate oxidase activity. Measured in this direction two problems arise. Since the equilibrium constant of the reaction pyruvate + NADH + H + = lactate + NAD + in phosphate buffer at pH 7.0 and 25°C is about 3 x 10 12 (Neilands 1952; Hakala et al. 1956) the reaction will proceed in the direction of lactate oxidation only when the concentration of NAD + and lactate are high. For the latter a value of at least 5 mM has been suggested (Everse and Kaplan 1975). When used for the determination of lactate it is customary to pull the reaction 'to the left' by trapping both H + and pyruvate with an alkaline buffer (usually glycine-NaOH, pH 9.0-10.0) containing hydrazine or semicarbazide (Bergmeyer 1974). Such a system is much less unphysiological
154 than it may appear at first, because a) neither hydrazine nor semicarbazide seem to have an effect on the initial velocity of the lactate oxidase reaction (Neilands 1952; Hakala et al. 1956; Winer and Schwert 1958); b) the pH optimum of the lactate oxidase reaction is around 9.0 (Winer and Schwert 1958); c) as in the living cell the accumulation of pyruvate is prevented. The last point is of particular relevance when the LDH isoforms studied are sensitive to pyruvate inhibition, as is the case with many isoenzymes of poikilotherms at low temperature. Pyruvate trapping systems have been used for establishing the kinetic constants of the lactate oxidase reaction (Hakala et al. 1956; Winer and Schwert 1958). More recent authors, on the other hand, appear to have been worried about what they considered the unphysiological nature of these assay conditions and have used more neutral buffers, usually Tris/HCI pH 7.4, without pyruvate trapping reagents (Hulbert and Moon 1978; Driedzic et al. 1985). In our hands these conditions led to rapid inhibition of some isoforms of fish LDH when the rate of lactate oxidation was measured. In consequence, and in view of what has been said above, we employed the following assay system for measuring the kinetic constants of the lactate oxidase reaction: Buffer: Dissolve 0.94 g glycine in 20 ml distilled water; set pH to 9.0 by adding 1.5-2.0 ml of a 24°7o solution of hydraziniumhydroxide (Merck); make up volume to 25 ml which gives a buffer concentration of 0.5 M. Assay: Buffer, NAD + to give an end concentration of 4.8 mM, varying concentrations of L-lactate (lithium salt), enzyme solution. The pH of the buffer was set at 20°C and left to vary with temperature, increasing to 9.11 at 4°C, decreasing to 8.93 at 24°C. This results in a dpH/dT(°C) value of 0.007 which is much lower than the 0.015-0.020 characteristic of imidazol buffers and the body fluids of ectothermic animals (Wilson 1977, Reeves 1977). Thus it may be assumed that under the conditions chosen both the reducing and the oxidizing reaction catalyzed by LDH proceeded at a more or less constant pH value, 7.5 and 9.0 respectively, over the range of experimental temperatures em-
Fig. . Starch gel electrophoresis od LDH isoenzymes from homogenates of red (R) and white (W) muscle of representatives of three families of freshwater fish, a coregonid (Coregonus sp.), a salmonid (Salvelinusfontinalis), and two cyprinids (Rutilus rutilus and Cyprinus carpio). S = start.
ployed. The kinetic constants were calculated by means of a programme based on the graphic method of Eisenthal and Cornish-Bowden (1974).
Results Isoenzyme patterns in red and white muscle from different species (Fig. I) Following the nomenclature suggested by Bailey et al. (1976) we shall designate the two major loci of LDH in fish muscle by LDH M and LDH H (A and B according to a different nomenclature), the basic set of five isoenzymes thus ranging from M4 to H4 . The recent duplication of loci observed in salmonids (and - as shown below - in coregonids) are designated Ma, Ms , H,, H, and the various alleles for loci which show polymorphism by capitalized Arabic superscripts, e.g. LDH H4Aand LDH H4B. In all species investigated the white muscles are characterized by the occurrence of only the most cathodic isoenzymes or group of isoenzymes. In cyprinids only M4 and , sometimes but always much more weakly expressed, M3HI, in salmonids and coregonids only the cathodic group Ma 4 to M4, are present. The red muscles contained either the
155
full set of isoenzymes (cyprinids) or a selection in which the anodic isoenzymes dominated. The most striking difference between the two types of muscles was met in Coregonus sp. in which the occurrence of the M-group and the H-group of isoenzymes was mutually exclusive. In salmonids the H-set was dominant in the red muscles but there were also members of the M-set and intermediate bands of the Ma Ha,, type present. This is in accordance with the findings of Bouck and Ball (1968). In C. carpio red muscle the H4 isoenzyme (in our population represented by two alleles, HA and HB) was always much more strongly expressed than the other isoenzymes, whereas in the red muscles of R. rutilus, L. cephalus, A. alburnusand S. erythrophthalmus the five isoenzymes occurred with more or less equal activity. In one population of R. rutilus (Seefeld) the H locus was represented by two alleles.
Table 1. Pyruvate affinities (Km values in mM) at 24°C of LDH in homogenates of red and white muscles, or of separated individual isoenzymes, in different species of fish. Km(pyr) (mM) at 24°C white muscles or M4
red muscles or H4
Rutilus rutilus (separated isoenzymes) (homogenate)
0.35 0.38 + 0.13
0.1 0.26 ± 0.06
Cyprinus carpio (homogenate)
0.48 + 0.08
0.1
Salvelinus fontinalis (homogenate) Coregonus sp. (homogenate)
0.14
0.95 0.89 ± 0.23
+ 0.03
0.3
+ 0.11
Either mean values + S.D. of three experiments, or the means of two experiments, are given.
Kinetic properties of lactate dehydrogenases Temperature profiles of pyruvate affinity. The cathodic isoenzyme(s) which dominate the white muscles of all species investigated displayed very similar temperature profiles of pyruvate affinity (Figs. 2-4). An increase in Km(pyr) from 4 to 24°C was observed in all species studied, although these differ widely in ecology and temperature preference. In salmonids the a and 3 loci of the M-group possess identical temperature relationships (Fig. 3), thus supporting the notion (Bailey et al. 1976) that this group arose through fairly recent duplication of the original M locus. The temperature profile of the most anodic isoenzyme (H4 ) or group of isoenzymes (h. 4 to H4), present only in red muscles, is also identical in all species of fish investigated. The temperature dependence of pyruvate affinity of this isoenzyme is generally weaker than that of the M isoenzyme but its major distinctive feature is its much lower Km value, the difference being most clearly expressed at the highest experimental temperatures. This is illustrated by comparing the two separated isoenzymes, or sets of isoenzymes, or the homogenates of red and white muscles from those species in
which each type of muscle is dominated by one type, or set, of isoenzyme(s) (Table 1). The latter condition holds for some salmonids and for Coregonus sp., but also for C. carpio in which species H4 was the dominant isoenzyme of the red muscle, whereas in the other four species of cyprinids all five isoenzymes occurred with about equal activity in this tissue (Fig. 1). In accordance with this distribution the temperature profile of pyruvate affinity is very similar in red muscle homogenates of the four temperate water cyprinids (Fig. 4), whereas it is characteristically different in the warm water species, C. carpio (Fig. 2). The same difference holds for the red muscle of the salmonid species investigated which display great variability both in isoenzyme distribution and in the temperature relationship of Km-values. Two extreme cases of the latter have been illustrated in Fig. 4. It is concluded that the temperature profile of pyruvate affinity in the red muscle of fish is mainly determined by the extent to which the cathodic isoenzyme(s) contribute(s) to total LDH activity. Substrate inhibition. By comparing the activities of LDH at high and low pyruvate concentrations one
156
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obtains a measure of the strength of substrate inhibition of this enzyme. When a quotient R is defined as the ratio of activities at 3.0 and 0.1 mM pyruvate concentrations, Fig. 4 attests to the very close rela-
tionship between Km and R values in all species and tissues examined. The higher pyruvate affinity the higher also the degree of substrate inhibition, i.e. the lower R. Thus highest affinity and strongest
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l
98R R7654321-
-6 .5 .4 .3 -2 .1 4 8 12 16 20 24
4 8 12 16 20 24
temperature (C) Fig. 4. Temperature profiles of Km, (mM pyruvate) and R values (ratio of enzyme activities at 3.0 and 0.1 mM pyruvate), in three families of freshwater fish, comprising data from 4 species of cyprinids, 5 species of salmonids, and one species of coregonid. Mean values and standard deviations are given. The salmonid data are summarized for white muscle homogenates only since the greater interspecific variability of isoenzyme composition of red muscle also implies greater variability of the temperature relationship of pyruvate affinity in this tissue (see text). The inset illustrates these differences by presenting the temperature profiles of pyruvate affinity in red muscle homogenates of Salmo gairdneriand Salmo truttafario.
159 Table 2. Ki values (mM lactate) of lactate oxidase reaction in red and white muscles of three species of freshwater fish measured at 2 temperatures. red muscle
Salvelinusfoninalis Coregonus sp. Rutilus rulilus
white muscle
4°C
20°C
4oC
20°C
11.0 + 1.3 4.9 + 0.8 6.0 + 1.9
7.5 + 0.8 3.4 + 0.5 2.5 + 0.5
14.7 + 0.8 12.3 + 0.9 14.8 + 1.4
10.7 1.0 8.5 + 1.7 3.7 + 0.9
Assay medium glycine-NaOH with hydrazine, pH 9.11 (4°C)-9.0 (20°C). Means and standard deviations of three independent samples.
inhibition is displayed by the cyprinid isoenzymes at low temperature, lowest affinity and zero inhibition by the salmonid and coregonid isoenzymes at high temperature. Lactate affinity. We measured the kinetics of the lactate oxidase reaction under alkaline conditions with pyruvate trapping at 4° and 20°C, in the red and white muscles of R. rutilus, S. fontinalis, and Coregonus sp. (Table 2). At 200 C the distribution of the Km-values for L-lactate corresponds to what was to be expected on the basis of the K,,-values for pyruvate; that is, substrate affinity is always higher in red than in white muscle fibres, the degree of difference reflecting the distribution of the isoenzymes in the muscle tissues of the three families investigated (Fig. 1). For example, the greatest difference in lactate affinity between red and white muscles occurs in Coregonus sp., the species with the greatest difference in the distribution of LDH isoenzymes between these tissues (Fig. 1). However, the results of the kinetic measurements at 4°C were totally unexpected and are at variance with the few data on the temperature relationship of the lactate oxidase reaction which we were able to locate in the literature (Henry and Ferguson 1985). Whereas in all species and tissues pyruvate reduction is characterized by a direct relationship between Km-value and temperature, the opposite is true for lactate oxidation - at least between 4° and
20°C (Table 2). Under all assay conditions tested (see Methods) the Km-value for lactate oxidation proved to be higher at 4 than at 20°C.
Discussion The distribution of LDH isoenzymes in red and white muscle fibres conforms to a well-known pattern (see Bouck and Ball 1968), the fast glycolytic fibres being characterized by the dominance of the most cathodic isoenzyme - or group of isoenzymes - whereas in the oxidative fibres a more varied pattern, always including the most anodic isoenzymes, prevails (Fig. 1). No attention seems to have been paid in the literature to the more subtle variations of this general pattern in different taxa of fish, and whether these varations might not enable us to draw biologically or physiologically meaningful conclusions. For example, there could be significance in the fact that in predatory species (coregonid, salmonids) the red muscle fibres are very clearly dominated by the anodic group of isoenzymes. In fact, in Coregonus sp., the most pronounced 'sprinter' of all species investigated (Hinterleitner et al. 1986), no overlap is observed between the isoenzymes of red and white muscle fibres, the former being characterized by an anodic, the latter by a cathodic, set of isoenzymes. In 'stayers', like the more slowly swimming cyprinids, the red muscle fibres are characterized by a more complete set of isoenzymes, and more moderate sprinters, like the salmonids, occupy an intermediate position in this respect. Even within one family difference can be found. For example, in the carp, the isoenzyme patterns of red and white muscles are more clearly distinguished than in the four other cyprinids investigated (Fig. 1). On the strength of other findings we believe that a functional explanation for this difference will be found, perhaps connected with the
160 different temperature relationships of the species concerned. The most complete separation of isoenzymes between red and white muscles is due to the duplication of individual LDH loci which allows either of the original M and H loci to express a set of five isoenzymes (Lim et al. 1975; Bailey et al. 1976; Klar et al. 1979; DiMichele and Powers 1982; Panepucci et al. 1984). One could visualize this more recent process of duplication as a mechanism by which the tetramers required for LDH activity can be constructed from closely related subunits. For example, within the M group of S. fontinalis the a and 3 loci exhibit identical temperature profiles for pyruvate affinity, whereas the profiles of the old M and H loci differ greatly from each other (Fig. 3). Thus, if for functional reasons a homogeneous population of tetramers were required the M, MO series of isoenzymes would be more suitable than the M,H series. This is so because the more closely related series of isoenzymes would be able to carry out a specialized task without the additional intervention of posttranslational control (Whitt 1970; Rosenberg 1971; Vesell 1975). This may be the reason why the more recent duplications of the M and H loci occur preferentially in fast-swimming carnivorous fish in which the sustained and the burst-like mode of swimming activity are more clearly separated than in herbivores and omnivores. However, despite their more homogeneous substrate relationship new and subtle differences have already appeared between the isoenzymes of the more recently duplicated alleles of LDH (Lim et al. 1975; Kao and Farley 1978; Klar et al. 1979). In fact, a slight shift in expression of white muscle LDH isoenzymes in the direction of the more aerobic B' subunit (corresponding to our 3 subunit) was observed when rainbow trout were subjected to sustained swimming activity for 200 days (Davie et al. 1986). In order to characterize the kinetic differences between the LDH's of red and white muscle fibres we used experimental temperature as an analytical tool (Figs. 2-4). We did not intend a detailed study of the temperature relationships of substrate affinities since for this purpose the temperature regimes of the environments and of the holding tanks were
not sufficiently well known or controlled. However, it should be pointed out that the up to threefold difference in pyruvate affinity between cyprinids on the one hand, salmonids and coregonids on the other hand (Fig. 4), is difficult to interpret in terms of the 'stable Km hypothesis' advanced by Yancey and Somero (1978). These authors held that in fish from a wide variety of habitats the K(pyr) values are regulated between 0.2 and 0.3 mM at the approximate range of temperature of the species. This conclusion is at variance with the large difference in Km-values between sympatric species (for example R. rutius, Coregonus sp., S. gairdneri which co-occur in many Austrian lakes). On the other hand, two cyprinids with very different temperature relationships, C. carpio and R. rutilus, display the same temperature profile of pyruvate affinity (Fig. 2). Rather it appears that differences in the substrate affinity of LDH reflect differences in the metabolism of swimming muscles. This could account for the kinetic differences not only between red and white fibres but also between the LDH's of the three families of fish studied. For example, in cyprinids lactate production is turned on at lower fractions of maximum swimming speeds than in salmonids (Driedzic and Kiceniuk 1976; Jones 1982; Wieser et al. 1986). Thus the LDH of cyprinid muscles should act as a lactate oxidase at lower concentrations of lactate than the LDH of salmonid muscles. This is borne out by the Km values of the lactate oxidase reaction in red muscles which are about three times higher in S. fontinalis than in R. rutilus (Table 2). One would have expected the coregonid species to resemble the salmonid in this respect, but its Km value for lactate oxidation is only slightly higher than that of R. rutilus red muscle. On the other hand, the white muscles of Coregonus sp. behave, as far as their affinity for lactate is concerned, like those of a salmonid species. There may be a biological message hidden in these differences. The Km values of lactate oxidation, as determined by our assay method, are in the range to be expected from the concentrations of lactate in fish blood and muscle after strenuous exercise. Whenever sampling artifacts are avoided by applying the correct procedure of freeze clamping (Wieser et al.
161 1986) concentrations of not more than 20 ptmol lactate per gram fresh weight of muscle, or ml of blood, are found both in salmonids (Black 1957; Driedzic and Kiceniuk 1976) and in cyprinids (Driedzic and Hochachka 1976; Wieser et al. 1986). This agrees well with Km values of from 2.5 + 0.5 to 7.3 + 0.9 mM lactate for the red muscle LDH's of our species measured at 20°C (Table 2). Km values exceeding 20 mM lactate, as reported, e.g., by Driedzic et al. (1985) for the LDH's of fish hearts would appear to be artifacts due to the assay method used by these authors. We have no explanation for the temperature anomaly of lactate affinity of the LDH's in our preparations as summarized in Table 2. If this finding reflects the conditions in vivo then the temperature relationship of pyruvate reduction must be different from that of lactate oxidation, with the consequence that the rate of lactate production during strenuous exercise may be unaffected by low temperature, whereas recovery should be slowed down by this factor. The former supposition is almost certainly correct (Brett 1964; Wieser et al. 1985), for the latter we have no evidence. At any rate, the difference in temperature relationship between the pyruvate reducing and the lactate oxidizing reaction cannot be due to differences in the pH/T relationship of the two assay media employed since the dpH/dT value was practically zero for both reactions (see Materials and methods). In conclusion it can be said that the properties of LDH in the red muscles of teleosts strongly indicate that this tissue plays an important role in the oxidation of lactate produced during locomotory activity in the white muscles, and that the extent of this ability depends on the biology of the species in question, as well as on temperature and other environmental factors.
Acknowledgement This research was supported by the 'Fonds zur Forderung der wissenschaftlichen Forschung in Osterreich', project no. S-35/04.
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