Biologia 64/1: 192—196, 2009Section Cellular and Molecular BiologyDOI: 10.2478/s11756-009-0017-7

Functional and adaptive significance of differentially expressedlactate dehydrogenase isoenzymes in tissues of four obligatoryair-breathing Channa species

Riaz Ahmad

Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh – 202 002 (U.P),India; e-mail: riazzool@rediffmail.com

Abstract: This study investigates the differential expression of lactate dehydrogenase (LDH) isoenzymes in the genusChanna using PAGE. With the help of obligate air-breathing, all of the selected species can sustain water deprivation tovarying degrees. In subunit composition and higher electrophoretic mobility of LDH-A4, the profiles of channid specieswere similar to other teleosts documented in the literature. However, inter- and intra-species differences, with particularreference to aerobic/anaerobic metabolic options, existed. Whereas glycolysis in Channa punctata appears to depend largelyon aerobic LDH-B and partly on anaerobic LDH-A, metabolism in C. gachua, C. striata and C. marulius depends exclusivelyon the activity of anaerobic LDH-A. Expression of the third locus Ldh-C was recorded in the eyes of C. marulius, in additionto C. gachua. Heat inactivation experiments reveal species differences between LDH isoenzymes and a general order of therelative stabilities: LDH-C > LDH-B > LDH-A. Metabolic and evolutionary implications of the findings have also beendiscussed.

Key words: LDH isoenzymes; LDH-C; Channa; differential expression; steady-state patterns; thermal inactivation; adap-tive significance.

Abbreviations: LDH, lactate dehydrogenase; ABO, air-breathing organ; kd, rate constant of inactivation.

Introduction

Sensitivity of lactate dehydrogenase (LDH; EC 1.1.1.27)and other enzymes to environmental adaptations is anarea of current interest (Ahmad & Hasnain 2005, 2006;Lushchak et al. 2005; Bagnyukova et al. 2006). Temper-ature and oxygen availability are among predominantenvironmental determinants of evolutionary as well asphysiological adaptation in teleost fishes (Hochachka1980; Das & Ratha 1996; Pierce & Crawford 1997;Somero 2004). LDH activity or LDH isoenzymes areone of the best indicators of switch-over to anaerobicmetabolism by which teleosts survive periods of oxygenunavailability (Chippari-Gomes et al. 2003). However,mostly the behavioral air-breathers have been the sub-ject of stress studies, while information is scanty onair-breathing teleosts which possess air-breathing or-gans (ABOs).This report deals with specificity of tissue expres-

sion of four species of genus Channa, which are ofcommon occurrence in India. Species of genus Channaare obligatory air-breathers equipped with accessorypouch-like ABO, the suprabranchial chamber (Mun-shi 1962; Graham 1997). Thus, their bimodal breath-ing is not at par with the physiology of behavioral air-breathers or the species which depress their metabolicdemands when oxygen is low (Dalla Via et al. 1994).

Channids dwell similar natural habitat despite theirwide distribution ranging from mainland China upto Iran, covering the entire Southeast Asian countriesand even southern Russia (Berra 2001; Musikasinthorn2003). Species variations in survivability during wa-ter deprivation are another important characteristicof the four Channa species, selected in present workfor LDH-PAGE profiling. Thus they are good mod-els to investigate if the steady-state expressions of tis-sue LDH isoenzymes during bimodal air-breathing dis-play species variations. It is well established that unlikemammals, physiological restrictions in fish tissues bringdown the number of LDH isoenzymes to less than five(Masters & Holmes 1975). Our previous study showedthat to meet energy requirements, Channa punctata re-sorts to the activation of anaerobic pathway that is re-flected in raised levels of LDH-A isoenzyme (Ahmad &Hasnain 2005).

Material and methods

Procurement of fish speciesLive, mature specimens of Channa punctata, C. gachua, C.striata and C. marulius were brought to laboratory fromthe local fish market. Following their identification, the fishspecies were acclimatized in tap water tanks for two dayswith freedom to gulp air. They were fed with commercial

c©2009 Institute of Molecular Biology, Slovak Academy of Sciences

Lactate dehydrogenase isoenzymes in genus Channa 193

diet. The dissolved oxygen was estimated using the Win-kler’s titrimetric method as described in APHA (1989). Inthe randomly sampled tap water, the dissolved oxygen was6.4 ± 0.5 ppm under laboratory conditions (25 ± 2◦C).

Preparation of tissue extractAdult fish were stunned by a head-blow, pithed and decap-itated. Brain, eye, heart, liver, anterio-dorsal white skele-tal muscle and kidney tissues biopsies were taken and eachsample was homogenized in pre-weighed vials containing 3–5mL ice-cold 50 mM Tris-HCl buffer (pH 7.5) as described byAhmad & Hasnain (2005). The clear supernatant obtainedafter centrifugation of homogenates at 4◦C and 15,000 rpmfor 20 min was taken for analysis using PAGE. Five sets ofexperiments containing five fishes each were done for everyselected species. All experiments were carried out follow-ing guidelines of the University Ethical Committee (AligarhMuslim University, Aligarh, India).

Protein estimationProtein concentration in the supernatant of different tissueextracts of selected fish species was measured following themethod of Lowry et al. (1951) using the BSA as the stan-dard.

Thermal inactivationPreliminary experiments were carried out to perform thethermal inactivation of LDH isoenzymes. The tissue ex-tracts prepared in 50 mM Tris-HCl buffer (pH 7.5) con-taining 2 mg protein/mL were incubated at a temperatureof 50◦C for predetermined time intervals. The inactivationwas stopped by transferring the container tubes to crushedice. Following centrifugation at 12,000 rpm and 4◦C for 20min, supernatant was collected and monitored on PAGE.

Electrophoresis and visualization of LDH isoenzyme bandsElectrophoresis was performed on 7.5% polyacrylamide gelsin vertical slabs (10.0 × 9.4 × 0.01 cm) and a modifieddiscontinuous system as described earlier (Ahmad & Has-nain 2005). To avoid distortions and smearing effects inLDH isoenzyme bands the samples were electrophoresed in1× upper gel buffer (pH 6.8) and later replaced with Tris-glycine buffer (pH 8.4) as the samples entered the lowerseparating gel. LDH isoenzymes were visualized in gels byhistochemical staining using L-lactate as substrate (Shaw& Prasad 1970). In staining mixture, nitro-blue-tetrazoliumserved as the hydrogen acceptor, while phenazine methosul-fate and β-NAD as the intermediate. Gels were incubatedat 37◦C for 30 min in the above staining mixture (pH 7.5).LDH isoenzyme activities were visualized as purple to darkblue bands in gels. Stained gels were fixed in 7% acetic acid(v/v) and documented by scanning on all-in-one HP Deskjet(F370) computer assembly.

Statistical analysisLDH-gel scans were processed through the software analy-ses. GelPro (Media Cybernetics, USA) was used for quan-titative analysis of LDH isoenzyme bands, while densito-metric profiling was carried out by Scion Imaging software(Beta release-4, Scion Corporation). Activities of individualbands in a PAGE lane was summed up and taken as 100%activity. Activity of an individual isoenzyme band was ex-pressed as the percentage of the total 100% activity andis represented as bars. Due to small sample size (n = 5),Student’s t-test was applied and the differences in the LDHtetramers activity were considered significant at P < 0.05.

Results

Figure 1 displays typical LDH zymograms of selectedtissues of Channa species during bimodal breathing.The subunit composition was determined by heat in-activation experiments. Under the electrophoretic con-ditions applied in this study, no interspecies differ-ences in the relative electrophoretic mobilities of A4or B4 LDH tetramers were recorded (Fig. 1). Substan-tial interspecies differences in the tissue distributionof LDH isoenzymes are, however, apparent. While inwhite skeletal muscle of C. punctata LDH-A4 is themajor activity, LDH-B4 or B subunit-rich heterote-tramers predominate in its eye, heart, liver and kidney(Fig. 1A,B,C). In contrast, LDH-A4 or A subunit-richheterotetramers are the major activities in tissue zymo-grams of the other three species (C. gachua, C. striataand C. marulius). In the heart of C. gachua, LDH-B4was detected along with traces of LDH-A4 and -A2B2.A remarkable character of LDH profiles of eye ex-

tracts of C. gachua and C. marulius is the expression ofa third locus, C (Fig. 1A – eye; lanes: Cg & Cm). Banddesignated C4 in LDH profiles of eye of C. maruliushas greater electrophoretic mobility than LDH-C4 of

Cp Cg Cs Cm Cp Cg Cs Cm

Fig. 1. Typical LDH zymograms of different tissues of adult chan-nid species. In frames A–C, lanes 1–4 for each tissue are: Channapunctata, C. gachua, C. striata and C. marulius, respectively.Symbols are: Cp = C. punctata, Cg = C. gachua, Cs = C. stri-ata, and Cm = C. marulius.

194 R. Ahmad

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Fig. 2. A comparison of quantitative differences in homo- and heterotetramers of LDH in different tissues of four Channa species:brain, eye, heart, liver, muscle, and kidney of investigated species of genus Channa. Symbols are the same as those in Figure 1. Relativeintensity of each band (LDH tetramer) in individual lanes is expressed as mean ± S.D. of five PAGE profiles. Mean values were plottedand considered significant at P < 0.05. Only negative values of standard deviation are plotted in the bars.

C. gachua eye. In C. marulius, heterotetramers LDH-B2C2 and -B1C3 can also be visualized in between B4and C4 (Fig. 1A). No such bands are identified in anyother tissue of C. gachua and C. marulius or eye ex-tracts of the C. punctata and C. striata.Percentage total LDH activities in individual lanes

of different tissues are plotted in Figure 2. Total LDHactivity is highest in the tissues of C. punctata (Fig. 2).Intraspecies qualitative variations in LDH profiles areprominently displayed by quantitative profiles of differ-ent tissues (Figs 1,2).Relative thermal stabilities of LDH homotetramers

in different tissues of C. punctata, C. gachua, C. stri-ata and C. marulius are compared in Figure 3. LDHactivity of -B4 of C. punctata and -C4 isoenzymes of

C. gachua and C. marulius is most thermostable andlasts up to 25 and 30 min following commencement ofincubation, respectively. Some intraspecies differencesin the relative thermostability are also apparent. Rateconstants of inactivation (kd) of LDH homotetramersderived from these first-order plots are listed in Table 1.According to the data, relative order of stabilities ofLDH homotetramers is: C4 > B4 > A4.

Discussion

So far as the relative electrophoretic mobility and sub-unit composition is concerned, LDH isoenzymes ofChanna species are similar to extensively documentedelectrophoretic profiles of other teleosts (Champion

Lactate dehydrogenase isoenzymes in genus Channa 195

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Fig. 3. Kinetics of inactivation of LDH homotetramers LDH-A4,LDH-B4 and LDH-C4 activity at 50◦C in selected fish species ofgenus Channa.

Table 1. Apparent rate constants (kd) of inactivation of LDHhomotetramers of different species of genus Channa calculatedfrom the first order kinetics plots of Figure 3.

Name of fish species Isoenzyme type kd values (s−1)

Channa punctata Cp LDH-A4 11.51 × 10−4Cp LDH-B4 6.60 × 10−4

Channa gachua Cg LDH-A4 10.83 × 10−4Cg LDH-C4 6.31 × 10−4

Channa striata Cs LDH-A4 9.04 × 10−4Channa marulius Cm LDH-A4 9.5 × 10−4

Cm LDH-C4 5.58 × 10−4

et al. 1975; Wright et al. 1975; Phillip et al. 1979;Whitt 1983; Allendorff et al. 1984). LDH-B tetramerof Channa species stacks near the sample slot, LDH-Ain between and LDH-C is the farthest migrating band(Fig. 1). Tissue distribution patterns of LDH isoen-zymes, which is determined either by the genetic andphysiological factors, are however specific. C. punctatais rich in LDH activity, because Ldh-A and -B loci areexpressed in all other tissues (Fig. 1). In white skeletalmuscle, the expression of LDH-B is extremely low. Theproportions of homo- and hetero-tetramers of anaer-obic LDH-A and aerobic LDH-B in C. punctata un-dergo adjustments during ontogeny or blocking the air-breathing (Ahmad & Hasnain 2005). Molecular data onbehavioral air-breather killifish suggests that regulatoryelements or regulatory genes may be involved in expres-sivity of structural genes of LDH or their physiologicalalterations (Schulte et al. 1997, 2000; Rees et al. 2001).Unlike C. punctata, a general preference for LDH-

A or heterotetramers rich in A subunit was displayed byC. gachua, C. striata and C. marulius (Figs 1,2). Thusas per tissue LDH profiles, metabolism in these threeChanna species is fueled by anaerobic glycolytic optionduring bimodal breathing. In terms of survivorship out-side water, when the fish exclusively depends on aerialrespirations,C. gachua is superior to C. punctata as wellas the other two species. C. gachua is also regarded as

the most generalized form from the evolutionary pointof view (Chandy 1955). It may thus be inferred thatexercising anaerobic option evolved as a key element oftheir survivorship.The third distinct Ldh locus LDH-C, initially dis-

covered as eye-specific E band, was subsequently re-ported in nearly all of the teleosts investigated so far(Markert & Faulhaber 1965; Whitt 1970; Markert et al.1975; Almieda-Val & Luis Val 1993). Earlier work onbiochemical and immunological characterizations hadestablished close similarity of LDH-C with LDH-B. Itwas suggested that a -B gene duplication event gaverise to LDH-C at about the time of origin of thebony fishes, approximately 350 million years ago (Whitt1969; Whitt et al. 1973; Markert et al. 1975). An al-ternative view that proposes closeness between LDH-Awith -C genes has been put forth by Li & Tsoi (2002).Their proposal is based on neighbor joining analysisof 49 nucleotide sequences of LDH isoenzyme genes ofvertebrate. A more detailed account of divergence thatspecifically deals with channids has recently been re-ported by Li et al. (2006). Heat stability data on chan-nids does not provide any insight to extend this dis-cussion to their origin any further. The data, however,suggests structural differences between LDH-A, LDH-B and LDH-C at species level (Fig. 3, Table 1), thoughno interspecies differences are observed in their relativeelectrophoretic mobilities.The data presented in this report suggests that

LDH isoenzyme patterns of homologous tissues maysubstantially vary among congeneric fish species de-spite sharing the same ambience and similar respira-tory physiology. Apparently, there are two classes ofsuch variations in distribution of isoenzymes: (1) thosewhere subunit restriction leads to preferential combi-nation or association of LDH subunits (McGovern &Tracy 1981; Fields et al. 1989; Coquelle et al. 2007); or(2) those that have restricted tissue expressions, suchas the expression of LDH-C in the eye of C. gachua(Murphy & Crabtree 1985; Murphy et al. 1990). Theformer class is under the influence of some adaptivestrategy based on their metabolic demands, aerobic oranaerobic.

Acknowledgements

The author is thankful to the Chairman, Department of Zo-ology for providing the necessary facilities. Thanks are alsodue to Dr. Absar-ul Hasnain for his valuable suggestionsand Dr. A. L. Bilgrami, Department of Mosquito Control,New Jersey, USA for kind cooperation. Research grant tothe author as SERC-Fast Track Scheme for Young Scien-tist (SR/FT/L-20/2005) from the Department of Scienceand Technology (DST), Ministry of Science and Technol-ogy, New Delhi is also thankfully acknowledged.

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Received February 28, 2008Accepted September 16, 2008