Thermal stability of soluble malate dehydrogenase isozymes of subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes

Thermal stability of soluble malate dehydrogenase isozymes of subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes

Maria do Carmo Monteiro¹, Maria Luiza B. Schwantes²,

Arno Rudi Schwantes² and Maria Regina de Aquino Silva²

¹Departamento de Ciências Fisiológicas, Faculdade de Medicina de Marília, Caixa Postal 451, 17519-030 Marília, SP, Brasil; ²Departamento de Genética e Evolução, Universidade Federal de São Carlos, Caixa Postal 676, 13565-905 São Carlos, SP, Brasil. Fax : [phone]. Send correspondence to M.L.B.S.

ABSTRACT

Electrophoretic thermostability tests of soluble malate dehydrogenases (sMDH) isozymes in tissue extracts of 21 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes showed three distinct results. The first, characterized by thermal stability of the slowest-migrating band or A-isoform, was detected in 52% of all species. The second, exhibited in 29% of the species analyzed, had a bidirectionally divergent pattern of their sMDH locus expression, and was characterized by a nondivergent thermostability pattern of both sMDH-A* and B*. In the third category, obtained in 19% of the species studied (the four Siluriformes species), thermostability of the fastest-migrating bands, or B-isoforms, was observed. Comparison of the effects of habitat temperature on the activity of paralogous and orthologous isoforms in tissue extracts of two of these species with different thermostability properties (Leporinus friderici - thermostable sMDH-A*, and Pimelodus maculatus - reverse thermostability properties or reverse electrophoretic pattern), collected during winter and summer months, showed that A and B subunits were present at different quantitative levels and their activities were nearly season independent. Differences in susceptibility to temperature (50°C) of both sMDH loci from tissue extracts of these species were found. In P. maculatus, these susceptibilities helped strengthen one of the hypotheses: the reverse thermostability pattern, where the fastest-migrating band or the B-isoform was the thermostable sMDH. Thus, temperature differences among orthologous homologues of sMDH seem to have occurred in these acclimatized species, where the fastest-migrating band, usually muscle specific and thermolabile in most teleosts, appeared in P. maculatus as the thermostable isoform.

INTRODUCTION

Adaptation to temperature by the two soluble MDH (sMDH: EC 1.1.1.37: malate: NAD⁺ oxidoreductase) gene loci of teleost fish where the sMDH-A* encodes a thermostable isoform and the sMDH-B* encodes a thermolabile one was first shown by Schwantes and Schwantes (1982a,b). Monteiro et al. (1991) studied sMDH of twenty-two subtropical fish species belonging to the orders Characiformes, Siluriformes and Perciformes, most of which showed the same duplicated gene loci. As in the case of other fishes and amphibians (Bailey et al., 1969; Wheat et al., 1971; Whitt et al., 1973; Schwantes and Schwantes, 1977, 1982a,b; De Luca et al., 1983; Coppes et al., 1987a; Fenerich-Verani et al., 1990; Aquino-Silva, 1992; Farias and Almeida-Val, 1992; Lin and Somero, 1995a,b; Caraciolo et al., 1996), sMDH in twenty of these species appeared as two or three anodal bands constituted by the association of subunits encoded by those gene loci. However, as well as other teleosts (Whitt, 1970; Bailey et al., 1970; Wheat et al., 1971; Farias and Almeida-Val, 1992), two of these species showed a much more complex electrophoretic pattern. In order to explain a six-band pattern detected in the characiform Hoplias malabaricus (Erythrinidae), an sMDH-A1,2* isoloci was proposed (Monteiro et al., 1991). In addition, the complex electrophoretic pattern detected in 87% of the perciform Geophagus brasiliensis (Cichlidae) specimens analyzed suggested three hypotheses: duplication in processing at sMDH-B*; presence of three loci, sMDH-A*, B1* and B2*, with a null allele within MDH-B2*, and overdominance (Monteiro et al., 1991).

Graves and Somero (1982), studying A4-lactate dehydrogenases of congeneric barracudas from the eastern Pacific, showed that differences in average or maximal habitat temperatures of a few degrees Celsius appear to be sufficient to favor selection for adaptively different orthologous enzyme homologues (homologues encoded by a single gene locus common to different species). Also, differential expression of paralogous protein homologues (homologues encoded by different gene loci in a species) during thermal acclimation has been reported (Hochachka, 1965; Hochachka and Somero, 1973, 1984; Tsukuda and Ohsawa, 1974; Tsugawa, 1976; Schwantes and Schwantes, 1982a,b; De Luca et al., 1983; Coppes et al., 1987 a,b,c; Coppes and Somero, 1990; Lin and Somero, 1995a,b). Schwantes and Schwantes (1982a), studying sMDH of several vertebrates, showed that in perciform Leiostomus xanthurus (Sciaenidae) tissue extracts, the paralogous isoforms were present at differing quantitative levels and their activities could be modified by changes in environmental temperatures. Furthermore, thermostability and thermal dependency tests showed that, similar to what occurs during acclimation, the A-isoform was more stable in the presence of heat than the B (Schwantes and Schwantes, 1982b). This latter appeared to be activated by low temperatures and thermolabile at high ones. De Luca et al. (1983), studying the subtropical fish Astyanax fasciatus (Characidae), also reported that A and B subunits were present at differing quantitative levels, but their activities were almost season independent. Lin and Somero (1995b), comparing sMDH of eastern Pacific barracuda from different latitudes, suggested that the variation in the ratio of paralogous thermostable and thermolabile sMDHs is important in adaptation to temperature, and that the absence of thermolabile isoforms in warm-adapted species may be a specific adaptation to high temperature. However, Farias and Almeida-Val (1992) and Caraciolo et al. (1996), studying the sMDH of Amazon fishes, detected in both forms a unidirectionally divergent expression pattern (predominance of thermostable-isoforms).

To examine these temperature-adaptive differences among paralogous and orthologous sMDH homologues, the present study describes MDH thermal sensitivities of 21 subtropical fishes from the southwestern region of Brazil captured during different seasons of the year.

MATERIAL AND METHODS

Animals

One hundred and eighty fish specimens from 21 species were collected from 4 reservoirs (Cachoeira de Emas, Ilha Solteira, Lobo, and Monjolinho) in São Paulo State and from 2 lakes (Carioca and D'Helvécio) in Minas Gerais State, Brazil. The fishes were caught with 2.5- and 3.5-cm mesh gill nets. Sampling data including orders, families, species, location, and number of specimens collected are given in Table I. Leporinus friderici and Pimelodus maculatus were obtained during different seasons (summer months - December to February and winter months - June to August) from Cachoeira de Emas and Monjolinho reservoirs. Habitat temperatures ranged from 16 to 18°C in winter and from 24 to 28°C in summer.

Table I
- Characteristics of sMDH from 21 species of subtropical fishes.

Family: Order Species	Reservoirs* and Lakes*	Thermal stability of sMDH loci
Anostomidae: Characiformes
Leporinus friderici (32)	A	A > B
L. steindachneri (10)	*	A = B
L. elongatus (8)	A	A = B
L. obtusidens (28)	A	A = B
L. octofasciatus (4)	C	A = B
L. steindachneri (10)	*	A = B
L. striatus (15)	A	A > B
Schizodon nasutus (13)	A	A > B
S. borelli (18)	B	A = B
Erythrinidae: Characiformes
Hoplias malabaricus (19)	D	A1 = A2 > B
Characidae: Characiformes
Salminus hilari (14)	A	A > B
S. maxillosus (4)	A	A > B
Serrasalmus spilopleura (20)	A	A > B
Brycon sp. (8)	*	A > B
Parodontidae: Characiformes
Apareiodon affinis (11)	A	A > B
Parodon tortuosus (7)	A	A > B
Pimelodidae: Siluriformes
Pimelodus maculatus (31)	A	A < B
Pimelodus sp. (13)	A	A < B
Pimelodela gracilis (14)	A	A < B
Loricariidae: Siluriformes
Hypostomus regani (17)	A	A < B
Cichlidae: Perciformes
Geophagus brasiliensis (15)	D	A = B1 = B2
Tilapia rendalli (17)	D	A > B

*Minas Gerais State (Carioca and D'Helvécio Lakes); D São Paulo State: A, Cachoeira de Emas; B, Ilha Solteira; C, Lobo and D, Monjolinho. Numbers within parentheses show individuals screened for thermostability tests.

sMDH preparation

Different tissue samples (skeletal muscle, heart, and liver) from each individual were dissected immediately after capture, frozen at -20°C, and homogenized (w/v) in 0.05 M phosphate buffer, pH 7.0, using a Potter-Elvejhem tissue grinder, at ice-cold temperature and then centrifuged at 19,000 g for 30 min at 4°C. The resulting crude extracts were utilized for electrophoresis and thermostability tests.

Electrophoresis

Electrophoresis was carried out in horizontal gels containing 14% (w/v) corn starch prepared according to Val et al. (1981), using the pH 6.9 Whitt (1970) buffer system. A voltage gradient of 5 V/cm was applied for 12-15 h at 4°C. After electrophoresis, the starch gels were sliced lengthwise and the lower halves were incubated in the dark, in an MDH staining solution: 4.3 x 10^-4 M NAD, 8.5 x 10^-4 M MTT, 1.6 x 10^-4 M PMS, in 0.2 M malic acid and 0.05 M Tris, pH 10.0, and agar 1.5%.

Nomenclature of sMDH gene loci, subunits and iso/allozymes is according to Shaklee et al. (1989).

Thermostability of sMDH

Temperature effect on the relative activities of sMDH subunits was tested by submitting 1 ml skeletal and heart extracts from each of the 21 species studied at 50°C for 1 and 2 min, 55°C for 1, 2 and 3 min, and 60°C for 30 s, 1, 2 and 3 min. Afterwards, these extracts were cooled on ice, centrifuged at 19,000 g for 30 min at 4°C, and electrophoresed side by side with the control. Controls were kept in an ice-water mixture. The same procedure was utilized to examine products expressed by the MDH-A1,2* isoloci of H. malabaricus liver extracts. Skeletal and heart extracts from L. friderici (12 summer and 15 winter specimens) and P. maculatus (18 summer and 11 winter specimens) captured during two seasons of the year were directly submitted to electrophoresis. After staining, the zymograms were made transparent by a drying process; therefore, it was possible to submit the bands to densitometric analyses. Malate dehydrogenase isozyme ratios were determined by scanning each gel on a Technolab Densitometer (520-nm filter) connected to a microcomputer with an analogic/digital interface and specific software. Results of the relative band concentrations and the corresponding graphics were obtained.

The effect of temperature was also measured spectrophotometrically in the direction of oxaloacetate reduction according to Schwantes and Schwantes (1982a). The tests involved the incubation of 1 ml of each extract (skeletal and heart extracts of L. friderici and P. maculatus) at 50°C in a water bath for 10-60 min, without substrate or coenzyme. Then, their activities were examined at 25°C. Afterwards, each extract was cooled, and MDH activity assayed by measuring the change in absorbance at 340 nm in a Beckman 25K spectrophotometer equipped with a Lauda K-2/R constant temperature circulating water bath set at 25°C. The assays were carried out in a 0.1 M potassium phosphate buffer, pH 7.0, containing 1.3 x 10^-4 M NADH and 3.3 x 10^-4 M oxaloacetate. All reactions were carried out in triplicate and were initiated by the addition of a small amount (10-50 µl) of extracts to the reaction mixture.

RESULTS

During the 50°C incubation no differences were detected among the 21 species analyzed; however, three types of patterns or interspecific diferences in thermal stability were noted when sMDH was incubated at 55°C and 60°C (Table I). The first pattern was characterized by the thermostability of the slowest-migrating bands, probably sMDH-A* products, in 52% of all species analyzed (the anostomids L. friderici, L. striatus and Schizodon nasutus; the erytrinid H. malabaricus; the characids Salminus hilarii, S. maxillosus, Serrasalmus spilopleura and Brycon sp.; the parodontids Apareiodon affinis and Parodon tortuosus; the cichlid T. rendalli) (Figure 1A). In H. malabaricus both A-isoforms exhibited a nondivergent pattern of expression among its different tissues, and thermostability tests also resulted in a nondivergent pattern (Figure 1B). In the anostomid L. friderici (Characiformes) two additional phenotypes were controlled by two slower alleles, *75 at the sMDH-A* (A100/75) and *88 at the B* locus (B100/88). Susceptibility of their isozymes and allozymes to temperature also suggested thermostability of MDH-A* alleles (*100 and *75).

The second, nondivergent pattern of thermostability was observed among paralogous isoforms in 29% of the analyzed species (the anostomids Leporinus steindachneri, L. elongatus, L. obtusidens, L. octofasciatus and S. borelli; the cichlid G. brasiliensis) (Figure 1C). In G. brasiliensis, a 6-band pattern was detected in 87% of the analyzed specimens. Thermostability tests showed no differences between the products detected in the MDH-B* region, nor between these and the A* ones. The third thermostability pattern involved the fastest-migrating band in 19% of the species studied (all siluriforms: Pimelodus maculatus, P. sp., P. gracilis and H. regani; Figure 1D). In P. gracilis, three phenotypes suggested the presence of one allele at each locus (A*68 and B*80). Susceptibility of their isozymes and allozymes to temperature showed the thermostability of the most anodic components. These results could suggest thermostability of the B* alleles or sMDH reverse electrophoretic pattern in these species.

Figure 1
- Heat inactivation of MDH in (A) Schizodon nasutus heart (H), (B) Hoplias malabaricus liver (L), (C) Leporinus elongatus heart (H) and (D) Pimelodus maculatus heart (H) MDH. Controls (co) were kept in an ice-water mixture. Tissue extracts were submitted to 50°C (for 1 and 2 min), 55°C (for 1 to 3 min), and 60°C (for 30 s, 1, 2 and 3 min).

We compared the effects of habitat temperature on the activity of paralogous and orthologous isoforms as estimated from densitometer tracings of stained gels from tissue extracts of two species with different thermostability properties: L. friderici (thermostable sMDH-A*), and P. maculatus (reverse thermostability properties or reverse electrophoretic pattern), collected during the winter (habitat temperature range, 16-18°C) and summer (habitat temperature range, 24-28°C). Relative activity of each subunit in relation to total sMDH activity (slowest-migrating bands/fastest-migrating bands - sm / fm) in fresh skeletal muscle (1.5 and 1.9, in winter and summer months, respectively) and heart extracts (3.8 and 3.2) from L. friderici showed the predominance of the slowest-migrating or A* locus band over the fastest-migrating or B* band in both tissues regardless of the season, and a low divergence in their field acclimatization responses (Figure 2A). However, unlike the bidirectionally divergent pattern of expression of the sMDH locus products previously detected by Monteiro et al. (1991) in 64 individuals of P. maculatus, skeletal muscle and heart extracts of twenty-nine other specimens of this species exhibited predominance of their fastest-migrating sMDH (B-isoform) or reversibility of their electrophoretic pattern, and sm/fm ratios approximately constant in both seasons (0.3-0.4 in winter and 0.4-0.5 in summer months) (Figure 2B).

Figure 2
- Relative activities of the MDH loci of skeletal muscle (M) and heart (H) extracts from Leporinus friderici (A) and Pimelodus maculatus (B) collected during winter (water temperature, 16°-18°C) and summer (water temperature, 24°-28°C).

In order to verify the temperature stability of the MDH of these field acclimatized species, thermal inactivation analyses were performed with the same tissue extracts utilized in densitometric analyses. When winter and summer acclimatized L. friderici heart extracts were exposed to 50°C for 10-60 min, their MDH half-lives were 55 min and 32 min, respectively (Figure 3A and B). Winter and summer skeletal muscle extracts submitted to this same test showed MDH half-lives of 17 min and 15 min, respectively. On the other hand, for P. maculatus tissues, half-life values of 10 min and 20 min were obtained for winter and summer heart extracts and well over 1 h for winter and summer skeletal muscle extracts (Figure 3C and D).

Figure 3
- Residual activity of unfractionated MDH preparations from heart (A and C) and skeletal muscle extracts (B and D) of winter and summer Leporinus friderici (A and B) and Pimelodus maculatus (C and D) following incubation at 50°C. wh, sh = Winter and summer heart extracts; wm, sm = winter and summer skeletal muscle extracts.

DISCUSSION

Three categories describe the types of duplicated gene expression. These categories are designated nondivergent, unidirectionally divergent and bidirectionally divergent (Ferris and Whitt, 1979). In a previous work on the sMDH of 22 subtropical fish species (Monteiro et al., 1991), we found two of these categories - bidirectionally divergent in 87% and unidirectionally divergent in 13% of them. In the first category, B-isoforms predominated in skeletal muscle extracts while A-isoforms were found to some extent in all tissues studied, and often predominated in the liver. In the second, sMDH-A* was expressed in all tissues, predominating over the B*. Therefore, in the study of thermostability properties and thermal acclimatization of the sMDH of these species, both skeletal, heart, and, sometimes, liver extracts were utilized. Electrophoretic thermal susceptibilities of the sMDH isozymes from 21 of these 22 previously analyzed species showed three distinct patterns. The first, characterized by thermal stability of the slowest-migrating band or A-isoform, was detected in 52% of all species. This thermostable sMDH was also reported by Schwantes and Schwantes (1982a,b), De Luca et al. (1983), Coppes et al. (1987a), Farias and Almeida-Val (1992), Lin and Somero (1995a,b) and Caraciolo et al. (1996) in other teleosts. In the characiform H. malabaricus, where the isoforms encoded by the sMDH-A* loci showed more stability to heat than that encoded by the sMDH-B*, a nondivergent pattern of this response among A1 and A2 dimers was detected (Figure 1B). The nondivergent pattern of expression previously detected by us (Monteiro et al., 1991), and of the thermostability detected here, for these isozymes, reinforces the recent locus duplication hypothesis for the sMDH-A* of this species. Also, in both MDH phenotypes of L. friderici detected, the susceptibility of their isozymes and allozymes to high temperature suggested a stability of sMDH-A* alleles in a nondivergent pattern. These results would suggest that the structural differences electrophoretically detected between the *100 and *75 alleles at sMDH-A* are not implicated in their response to temperature. Of the eleven species included in this divergent pattern of thermostability (A > B), 91% of which were Characiformes, ten exhibited a bidirectionally divergent pattern of their sMDH locus expression. Only the perciform T. rendalii showed a unidirectionally divergent expression where the A-isoform predominated in every tissue analyzed. So, it might be possible that in these species thermostability occurred in the early evolution of structure, function and gene regulation of MDH-A*.

The second category, exhibited by 29% of species analyzed here, all with a bidirectionally divergent pattern of their sMDH locus expression, was characterized by a nondivergent thermostability pattern of both sMDH-A* and B*. In these species, the pattern of gene expression seems to have no relationship with differences in heat stability of Aand B-isoforms. In the third category, obtained in 19% of species studied (the four Siluriformes species), thermostability of the fastest-migrating bands, or B-isoforms, was observed. In the three sMDH phenotypes of P. gracilis produced by the activity of A*68 and B*80 alleles, the susceptibility to high temperature of sMDH isozymes and allozymes examined in skeletal and heart extracts also suggested the thermostability of sMHD-B* alleles (*100 and *80). A number of cases of polymorphism have been described for sMHD-B* with different geographic distribution. According to several authors (Bailey et al., 1969; Place and Powers, 1978; Powers and Place, 1978; Kirpichnikov and Muske, 1980; Hines et al., 1983), environment and temperature at different latitudes would seem to be at least partly responsible for the clinal variation in allelic frequencies of this locus.

While in our previous analysis (Monteiro et al., 1991) two of these species, P. gracilis (68 individuals) and H. regani (17), exhibited a unidirectionally divergent pattern of their sMDH locus expression, P. maculatus (64) and Pimelodus sp. (13) showed a bidirectionally divergent one. However, except for P. maculatus, in the other three species the heterodimer was not detected (Monteiro et al., 1991). The lack of binomial distribution of MDH isozymes in these three siluriform species, predominance of the fastest-migrating component together with their thermostability data, suggested two hypotheses: thermostability of the sMDH-B* alleles or a reverse electrophoretic pattern. Kinetic properties of sMDHs of a diversity of fishes, amphibians, reptiles and birds suggest that when only one or two sMDH components were present they resembled those of L. xanthurus thermostable sMDH (Schwantes and Schwantes, 1982a). Schwantes and Schwantes (1982a), studying the MDH of the siluriform Ictalurus nebulosus, detected only one component which suggested a single and thermostable sMDH locus. On the other hand, for lactate dehydrogenase, according to several authors (Whitt, 1969; Markert et al., 1975; Panepucci et al., 1984; Coppes et al., 1987c; Ferreira et al., 1991) a reverse pattern is not unusual, occurring in one third of fish species analyzed. Ferreira et al. (1991), studying LDH in 27 species of Amazon fish, also found a reverse electrophoretic thermostability for 2 siluriform species.

Higher lability of mMDH detected at all temperatures (mainly at 55°C and 60°C) in species examined could be used as a reliable means for distinguishing between both sMDH and mMDH forms. Thus, detection of polymorphism at the mMDH locus of T. rendalli (Cichlidae), with a slower allele C*63 in 29% of the population (Monteiro et al., 1991), was confirmed by the thermostability results obtained with the three studied phenotypes, C100, C100/63 and C63.

Previous research on rainbow trout LDH (Moon and Hochachka, 1971), and other isozyme systems in fish (Hochachka, 1965; Baldwin and Hochachka, 1970; Schwantes and Schwantes, 1982a,b; De Luca et al., 1983; Lin and Somero, 1995a,b), indicated that the expression of different enzyme variants can depend upon environmental parameters such as season and acclimation. Our results on analyzing sMDH of L. friderici, H. malabaricus and P. maculatus, like the ones on the temperate fish L. xanthurus (Perciformes) (Schwantes and Schwantes, 1982a,b) and the subtropical A. fasciatus (Characiformes) (De Luca et al., 1983), showed that A and B-subunits occurred at different levels in different tissues. However, similar to the results obtained on A. fasciatus, the quantitative level of sMDH subunits was shown to be almost independent of season, when average water temperature varied from 17°C (winter) to 26°C (summer). According to De Luca et al. (1983), this near season independence of both MDH subunits in these subtropical fishes could be explained by low environmental temperature fluctuation. On the other hand, the experimental organism used previously by Schwantes and Schwantes (1982a) was an estuarine fish whose environment is characterized by varied water temperatures (often ranging from 5 to 30°C annually). In this species, where subunit distribution seems to depend on the season, activity ratio varied from 1.33 in winter spots to 2.74 in summer spots (Schwantes and Schwantes, 1982b). Thus, this species has to cope with a fluctuating environment in which selection for ability to survive and function effectively over a wide range of environmental conditions would be expected to have been quite rigorous. Rapid temperature fluctuations of several degrees occur daily in the environment of this species throughout which its enzymes must remain functional (De Luca et al., 1983). However, it is well known that activity staining on native gels does not provide a quantitative measure of the relative amounts of protein present in different bands, and densitometric methods suffer from nonlinearity when staining intensity plateaus at high enzyme concentrations (Markert and Masui, 1969). Perhaps on account of these problems, susceptibility to temperature (50°C) of either winter or summer heart and skeletal muscle extracts utilized for densitometric analyses was not always confirmed by those results. MDH half-lives obtained from winter and summer acclimatized L. friderici heart extracts confirmed the predominance of A-subunits seen in densitometric tracings and reflect the thermostability obtained in electrophoresis. But when this test was applied to winter and summer acclimatized L. friderici skeletal muscle, their MDH half-lives did not confirm the predominance of A-subunits in this tissue obtained from the densitometric tracings of gels. They reflected the thermolability of their products and agreed with their electrophoretic thermostability properties and other authors' results (Schwantes and Schwantes, 1982a,b; De Luca et al., 1983; Lin and Somero, 1995a,b). On the other hand, the half-life values obtained for winter and summer skeletal muscle extracts of P. maculatus which were consistent with densitometric analyses and electrophoretic thermostability tests (predominance of the fastest and thermostable isoform) helped strengthen one of the hypotheses, the reverse thermostability pattern, where the fastest-migrating band or the B-isoform was the thermostable sMDH. But when this test was applied to their winter and summer heart extracts (predominance of the fastest and thermostable isoform), their MDH did not show the same response detected for skeletal muscle, i.e., their half-lives did not confirm the predominant B-isoform as thermostable sMDH. Thus, temperature differences among orthologous homologues of sMDH seem to have occurred in these acclimatized species. Also, the idea of a thermostable B-isoform in the siluriform species studied here raises a question concerning paralogous and orthologous relations of sMDH loci intra- and interspecies, orders and classes.

Therefore, acclimatization temperature appears to influence in a different way the activity of both sMDH loci in tissues of the various species examined. Thus, the distinct responses of the paralogous homologues might be associated both with variations of modulation in their activities and their pattern of tissue expression. Crawford and Powers (1989, 1992), analyzing the LDH of the teleost Fundulus heteroclitus populations subjected to clinal temperatures that occur along the eastern coast of North America, verified that clinal variation according to temperature exists in the concentration of the heart - isoform. This compensatory change in LDH-B enzyme concentration was due to change in the amount of LDH-B mRNA via transcriptional regulation. Thus, evolutionary adaptation to different thermal environments via transcriptional regulation (Crawford and Powers, 1992) might also explain the different responses of sMDH-A* and B* in tissue extracts from the acclimatized species here analyzed.

ACKNOWLEDGMENTS

Research supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). The authors wish to thank Dr. Paula Ann Matvienko-Sikar, who critically reviewed this manuscript. Publication supported by FAPESP.

RESUMO

No presente trabalho, mostramos em ortólogos da sMDH de 21 espécies de peixes subtropicais das ordens Characiformes, Siluriformes e Perciformes, três diferentes estabilidades térmicas. A primeira, caracterizada pela termoestabilidade do componente menos anódico ou isoforma-A, foi detectada em 52% de todas as espécies. A segunda, exibida por 29% das espécies aqui analisadas, caracterizou-se por um padrão não-divergente de termoestabilidade dos locos sMDH-A* e sMDH-B*. Na terceira resposta, obtida em 19% das espécies analisadas (as 4 espécies Siluriformes), foi observada a termoestabilidade da banda mais anódica ou isoforma-B. O efeito da temperatura ambiental na atividade relativa de isoformas parálogas e ortólogas de duas dessas espécies com diferentes respostas térmicas (Leporinus friderici - sMDH-A*, termoestável, e Pimelodus maculatus, termoestabilidade reversa ou padrão eletroforético reverso), coletadas em meses de inverno e de verão, mostrou que as subunidades A e B estão presentes em seus extratos de tecidos em diferentes níveis quantitativos e suas atividades relativas são, praticamente, independentes da época de coleta. Na incubação de extratos de tecidos dessas 2 espécies a 50°C, diferentes respostas de inativação térmica foram dadas pelos locos da sMDH. Em P. maculatus, a resposta obtida em músculo esquelético ajudou a escolher a hipótese mais provável - a da termoestabilidade reversa, onde a banda mais anódica ou isoforma-B é a sMDH termoestável. Assim, diferenças na susceptibilidade à temperatura parecem ter ocorrido entre homólogos ortólogos da sMDH, nessas espécies, onde a banda mais rápida, normalmente músculo-específica e termolábil na maioria dos teleósteos, aparece em P. maculatus como a isoforma termoestável.

(Received February 24, 1997)

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Publication Dates

• Publication in this collection
  06 Jan 1999
• Date of issue
  June 1998

History

• Received
  24 Feb 1997