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Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita
Comparative Biochemistry and Physiology Part B 137 (2004) 197–207
Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita Anand S. Srivastavaa, Ichiro Ooharab, Tohru Suzukic, Steve Shenoudaa, Surender N. Singhd, Dharam P. Chauhana, Ewa Carriera,* a School of Medicine, University of California San Diego, La Jolla, CA 92093, USA National Research Institute of Fisheries Science, Fukuura 2-12-4 Kanazawa, Yokohama 236-8648, Japan c Metabolism Section, Fish Nutrition Division, National Research Institute of Aquaculture, Nansei Watarei, Mie, 516-01, Japan d Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi 221 005, India b
Received 25 July 2003; received in revised form 8 November 2003; accepted 10 November 2003
Abstract Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for L-alanine were 18.5"0.48 and 23.55"0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29"0.023 and 0.33"0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With L-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4=10y4 and 3.4=10y4 mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate Lalanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Alanine aminotransferase; Enzyme; Fish; GPT
1. Introduction Fish show remarkable capacity to utilize proteins and amino acids for both, energy production and biosynthetic processes (Renaud and Moon, 1980; *Corresponding author. Tel.: q1-858-822-1050; fax: q1858-822-0835. E-mail address: [email protected] (E. Carrier).
Suarez and Mommsen, 1987; Foster and Moon, 1991; Treberg et al., 2003; Mommsen et al., 2003). Obviously transamination reactions of amino acids are one of the critical steps in setting the pace of several metabolic events in fish. Alanine aminotransferase (AAT; EC: 2.6.1.2.), a pyridoxal-phosphate-dependent enzyme, has been observed to be one of the sensitive and key enzymes in this
1096-4959/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2003.11.006
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respect (Walton and Cowey, 1982). The enzyme, compartmentalized in cytosol (c-AAT) and mitochondria (m-AAT), is responsible for reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate. As a key enzyme that provides the substrate for the malate aspartate shuttle (Masola et al., 1985), this reaction becomes instrumental in transport of crucial metabolites across the mitochondrial membrane and thereby channeling alanine into general metabolic pathways. The profile of this enzyme has been shown to vary with age, sex and in a tissue-specific manner in several animals studied (Zinkel et al., 1971; Alexin and Papoutsoglou, 1986; Abraham and Rina, 1991). Extensive information on AAT is available from mammalian models. Crystallized cAAT from rat has been reported to contain two closely migrating sub-bands on PAGE (Matsuzawa and Segal, 1968), similar to those of the noncrystallized homogeneous enzyme (Gatehouse et al., 1967). The molecular mass of c-AAT was calculated to be 114 kDa from rat liver (Segal et al., 1962) and 90 and 115 kDa from pig heart (Milton et al., 1967a,b). Ishiguro et al. (1991) have worked out the complete amino acid sequence of the 55-kDa subunit of c-AAT from rat liver. AAT isolated and characterized from Drosophila nigromelanica was also shown to be a homodimer of 113 kDa (Chen, 1985). Extensive studies have been done on the metabolic importance of this enzyme in a number of mammals. In addition, AAT profiles have been suggested as one of the markers of a variety of viral and other infections in rat and human (Agarawal et al., 1996; Cahen et al., 1996; Rodriguez et al., 1997). In fish, however, available information is restricted to the alterations in AAT activities as a function of environmental factors and dietary compositions (Alexin and Papoutsoglou, 1986; ¨ Jurss et al., 1987; Foster and Moon, 1991; Sunny et al., 2002; Treberg et al., 2003; Malbrouck et al., 2003; Samsonova et al., 2003). We have demonstrated that different food habits and respiratory patterns bring about significant adaptive changes in the activity of c-AAT and aspartate aminotransferase (AspT) in different tissues of Clarias batrachus and Labeo rohita (Srivastava et al., 1998a,b, 1999). To study the mechanism of such changes in AAT, we now intend to address the question, ‘How does AAT adapt at the protein level as a function of different metabolic demands?’ Also,
though AAT has been purified from mammals, Drosophila (Chen, 1985), Candida maltosa (Umemura et al., 1992), Trypanosoma (Zelada et al., 1996), etc. studies on purification and macromolecular properties of the enzyme have not been a focus in freshwater fish. In this paper we report on the purification, characterization and structure– function relationship of c-AAT from the liver of two economically important freshwater fish of south-eastern Asia, C. batrachus and L. rohita. C. batrachus is a carnivorous air-breathing catfish, with suprabranchial arborescent accessory airbreathing organ that functions much like a lung. The cyprinid L. rohita is an herbivorous waterbreathing fish. 2. Materials and methods 2.1. Animals Female freshwater teleosts C. batrachus and L. rohita were collected from River Ganga in Varanasi during June and July (spawning phase of fish). C. batrachus, weighing 70–75 g and 18–20 cm length, and L. rohita, 150 g and 20–22 cm, were killed at approximately 10.00–11.00 h to avoid the effect of circadian rhythmicity for all experiments. 2.2. Chemicals Analytical grade chemicals were used. Biochemicals used were purchased from Sigma Chemical Co., St. Louis, MO. DEAE-Sephacel and LMW calibration kits were obtained from Pharmacia Fine Chemicals. Calcium phosphate gel was prepared in our laboratory according to the method of Keilin and Hartree (1938). 2.3. Assay of AAT The activity of c-AAT was assayed spectrophotometrically following the method of Segal and Matsuzawa (1970). The reaction mixture consisted of potassium phosphate buffer, 0.1 M (pH 7.4); L-alanine, 0.2 M; lactate dehydrogenase, 50 unitsy ml; NADH, 5.6 mM; 2-oxoglutarate, 0.25 M. Rate of conversion of alanine to pyruvate was monitored by coupling the reaction with the formation of lactate from pyruvate in the presence of NADH at 25 8C. The reaction was started by addition of 2oxoglutarate, and the decrease in optical density
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was recorded at 340 nm. A unit of enzyme activity was defined as the amount of enzyme that converts 1 mmol of NADH per minute at 25 8C. 2.4. Purification of c-AAT Cytosolic alanine aminotransferase was purified following the methods of Gatehouse et al. (1967) with slight modifications. All steps were carried out at 4"2 8C unless otherwise indicated. Immediately after killing the fish the liver was removed and washed in ice-cold 0.69% NaCl solution. Tissue was homogenized (10% wyv) in ice-cold 0.25 M sucrose solution containing 1 mM EDTA. Homogenate was centrifuged at 15 000=g for 30 min. Supernatant was subjected to heat treatment at 55 8C followed by acid precipitation at pH 5.5 by 1 M acetic acid. Supernatant collected after centrifugation at 10 000=g was used for ammonium sulfate fractionation. The pellet collected after 45% ammonium sulfate precipitation was dissolved in a minimum volume of 10 mM potassium phosphate buffer, pH 5.7, containing 1 mM dithiothreitol (DTT). Sample was dialyzed overnight against the same buffer. Dialysate was cleared at 40 000=g for 3 h. The clear fraction was applied to a 15=2 cm DEAE-Sephacel column equilibrated with 10 mM potassium phosphate buffer, pH 5.7, containing 1 mM DTT. The column was developed in the equilibrating buffer at a flow rate of 0.9 mlymin. The enzyme was eluted by the application of the same buffer containing 0.025 mM Na2SO4. Multiple fractions were collected and assayed for enzyme activity. Fractions with more than 40% enzyme activity were pooled and used for calcium phosphate gel treatment. A calcium phosphate gel suspension was mixed with the total volume of pooled fractions in the ratio of 20 mgy ml. After 5 min of gentle mixing, the gel was removed by centrifugation at 10 000=g. This process was repeated three times using fresh gel suspension each time. The treated fraction was concentrated by 50% ammonium sulfate precipitation. The pellet obtained was dissolved in 2.0ml 0.1 M potassium phosphate buffer, pH 7.5, containing 0.1 M L-proline and 1 mM DTT. The solution was chilled and 6.0-ml cold acetone maintained at y20 8C was added slowly and the tube was centrifuged immediately at 0 8C. The pellet was then dissolved in 2.0-ml 0.1 M potassium phosphate buffer, pH 7.5, containing 0.1 M Lproline and 1 mM DTT and centrifuged. After
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55% ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.1 M potassium phosphate buffer, pH 7.5, containing 1 mM DTT and EDTA. The solution was centrifuged again and the cleared suspension was stored at 4 8C. This was treated as the purified enzyme preparation. 2.5. Analytical methods The protein concentrations of the samples were determined by the Folin method (Lowry et al., 1951). For molecular weight determination of liver c-AAT, 20-mg desalted protein samples along with MW markers were subjected to 10% SDS-PAGE following the method of Laemmli (1970). After electrophoresis, gels were stained in 0.1% Coomassie brilliant blue R-250 and photographed. 2.6. In vitro studies The Km value for L-alanine of purified liver cAAT was determined by monitoring the enzyme activity at different concentrations of L-alanine (10–100 mM). 2-Oxoglutarate was kept constant at a saturating concentration (0.3 M) in these assays. The Km value for 2-oxoglutarate was determined by measuring the enzyme activity at varying concentration of the substrate, keeping L-alanine at its saturation level (0.25 M). Lineweaver–Burk plots of the data were used to determine the Km values. The effect of aminooxyacetate on the forward reaction was observed separately with respect to both substrates. The enzyme samples were assayed as described earlier, at different concentrations of aminooxyacetate (AOAA). KI values were calculated from a double reciprocal plot of the data. To study the effect of temperature, suitably diluted enzymes were incubated separately at different temperatures (15–70 8C) for 10 min in the standard assay mixture, and remaining activity was determined at 25 8C as described in the text. The effect of pH was monitored by incubating the enzyme samples separately in assay mixtures consisting of phosphate buffers over a pH range of 5.5–9.5 for 10 min at 37 8C followed by enzyme assay at 25 8C. 2.7. Statistical analysis Averages and standard deviations were obtained from four values each time. Student’s t-test was used to test significances.
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Fig. 1. Elution profile of liver c-AAT of C. batrachus on DEAE-Sephacel column. Ammonium sulfate fractionated dialyzed sample was applied on the column and elution was made as described in the text.
Fig. 2. Elution profile of liver c-AAT of L. rohita on DEAESephacel column. Ammonium sulfate fractionated dialyzed sample was applied on the column and elution was made as described in the text.
3. Results with a 38% yield from C. batrachus. The enzyme was purified 120-fold from the liver of L. rohita with a 27% yield. Nevertheless, both the preparations were observed to be nearly homogenous on SDS-PAGE (Fig. 3). SDS-PAGE results also exhibit that the final preparations from both the fish contain two closely migrating c-AATs (A and B) of 54 and 56 kDa,
Figs. 1 and 2 show that the enzyme c-AAT from both C. batrachus and L. rohita is eluted from a DEAE-Sephacel column following a similar elution pattern. Summary of stepwise purification protocol is presented in Tables 1 and 2. Combining two batch separations after ion-exchange chromatography, the liver c-AAT was purified 202-fold Table 1 Summary of purification of liver c-AAT of C. batrachus Steps of purification
Volume (ml)
Total units
Specific activity
Fold of purification
% yield
(1) (2) (3) (4) (5) (6)
128 118 20 10 12 6
472 427 303 250 223 180
0.44 0.47 3.00 39.7 77.5 89.5
1.7 6.7 89.8 175 202
100 90 64 53 47 38
Cytosolic fraction (15 000=g) Heat extraction (NH4)2SO4 precipitation DEAE-Sephacel column Ca3(PO4)2 Gel Acetone precipitation
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Table 2 Summary of purification of liver c-AAT of L. rohita Steps of purification
Volume (ml)
Total units
Specific activity
Fold of purification
% yield
(1) (2) (3) (4) (5) (6)
125 116 18 10 12 5.5
683 507 413 309 259 82
0.41 0.53 1.8 26.2 44.4 50.0
– 1.3 4.2 59.5 107 120
100 74 60 45 38 27
Cytosolic fraction (15 000=g) Heat extraction (NH4)2SO4 precipitation DEAE-Sephacel column Ca3(PO4)2 Gel column Acetone precipitation
as determined on semilogarithmic plot (plot not shown) prepared against standard molecular weight marker proteins. No differences in MW of liver c-AATs between the two species were noted. 3.1. Temperature and pH studies Fig. 4 presents the percent activity of purified c-AATs incubated and assayed at different temperatures. The temperature maximum is observed to be 37 8C for liver c-AAT from both fish. Temperature profiles at lower temperatures appear to be unaltered with the enzyme from both fish, whereas higher temperature ranges show significant inactivation in the enzyme activity of C. batrachus. Profiles of liver c-AAT from both fish exhibit significant differences at lower pH ranges with pH optima of 7.5 (Fig. 5).
Fig. 3. Ten percentage SDS-PAGE of liver c-AAT fractions at various stages of purification. (1) Twenty microgram protein was loaded from crude homogenate, (2) ammonium sulfate fraction, (3) DEAE-Sephacel elute and (4) purified preparation. M, marker protein; C, C. batrachus; L, L. rohita. Final preparations from both the fish contain two closely migrating c-AATs (A and B) of 54 and 56 kDa, as determined on semilogarithmic plot (plot not shown) prepared against standard molecular weight marker proteins.
3.2. Kinetics Table 3 indicates that apparent kinetic constants Km for L-alanine are calculated to be 18.5"0.48 and 23.5"0.6 mM and for 2-oxoglutarate values are 0.29"0.023 and 0.33"0.028 mM with liver c-AAT purified from C. batrachus and L. rohita, respectively. In the presence of L-alanine, AOAA acts as competitive inhibitor with KI values of 6.4=10y4 and 3.8=10y4 mM for liver c-AAT from C. batrachus and L. rohita, respectively (Fig. 6). AOAA behaves as an uncompetitive inhibitor
Fig. 4. Effect of temperature on purified c-AATs from the liver of C. batrachus (q) and L. rohita (D). The activity observed at 37 8C was taken as 100% and mean value of four observations has been plotted on each point. Each value is presented with the form of mean"S.D. ***P-0.001; *P-0.05.
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Fig. 5. Effect of pH on purified c-AATs from the liver of C. batrachus (q) and L. rohita (D). The activity observed at pH 7.5 was taken as 100% and mean value of four observations has been plotted on each point. Each value is presented with the form of mean"S.D. ***P-0.001; *P-0.01; **P-0.05.
of the enzyme in the presence of 2-oxoglutarate with similar KI values (4.8=10y4 and 4.3=10y4 mM; Fig. 7). 4. Discussion Alterations in key metabolic enzymes are instrumental in mediating metabolic adaptive changes in cells under the influence of external and internal factors. Enzymes have tremendous capacity to adjust at the structural level in this respect. Therefore, studies on structure–function relationship of key enzymes are of special interest. Particularly in fish, AAT has been found to show significant alterations under the influence of environmental factors and dietary composition. These changes in AAT activity may be due to alterations in the concentration of AAT in the tissues (Alexin and ¨ Papoutsoglou, 1986; Jurss et al., 1987; Foster and Moon, 1991). We have observed that different tissues of air-breathing carnivorous fish show significant increases in its activity compared to nonair-breathing herbivorous fish (Srivastava et al., 1998a). To understand the mechanism of such changes in c-AAT at the protein level, the enzyme was purified from the liver of C. batrachus and L.
rohita. Since literature is not available on the isolation of AATs from freshwater fish, we followed the method reported in other animal models with modifications realized during standardization of the procedure. To avoid denaturation of the enzymes during the entire process, appropriate concentrations of DTT and EDTA were included in all solutions. The pH was monitored and maintained continuously during the ammonium sulfate precipitation; L-proline was added to protect the prosthetic group of the enzyme as reported earlier (Segal and Matsuzawa, 1970). Summary of the purification indicates a twostep ion-exchange chromatography and acetone fractionation results in isolation of liver c-AAT to electrophoretic homogeneity from both C. batrachus and L. rohita. Similar purification results have also been reported from other animals (Segal and Matsuzawa, 1970; Patnaik and Kanungo, 1976; Guelbenzu et al., 1991; Umemura et al., 1992). Separation of two c-AAT peaks on ionexchange chromatography, one in unbound and another in bound fractions, also corroborates earlier reports in other animals (Harding et al., 1961; Chen and Giblett, 1971; Patnaik and Kanungo, 1976; Umemura et al., 1992, 1994). SDS-PAGE of the final preparation from the second peak of the liver c-AATs revealed two closely migrating protein bands of 54 and 56 kDa. If it is assumed that the native protein is a heterodimer of 110 kDa in the case of both fish, values are close to cAATs from pig cardiac muscle, rat liver (Milton et al., 1967a,b), human liver (Ishiguro et al., 1991) and Chlaymydomonas reinharditii (Guelbenzu et al., 1991). From our laboratory, another enzyme, glutamine synthetase from the brain of C. batrachus, has also been reported to be structurally close to the enzyme from mammals (Singh and Singh, 1992). Temperature and pH profiles of purified liver c-AATs from both fish are almost the same. The pH optima corroborate the earlier Table 3 Apparent Km values of purified liver c-AATs of C. batrachus and L. rohita with L-alanine and 2-oxoglutarate Substrate Fish
L-alanine
Km (mM)
2-oxoglutarate Km (mM)
C. batrachus L. rohita
18.50"0.48 23.55"0.60***
0.29"0.023 0.33"0.028
*** P-0.001 represents the comparison between two species. Mean"S.D. (ns4).
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Fig. 6. Double reciprocal plots for determination of KI for AOAA using L-alanine as substrate with the purified liver c-AATs from (A) C. batrachus and (B) L. rohita. Each point in the plot represents mean value of four observations.
reports from other vertebrates (Segal et al., 1962; Matsuzawa and Segal, 1968; Galichowski et al., 1977). Thus, it appears like glutamine synthetase (Singh and Singh, 1992), functionally c-AAT has not undergone much evolutionary change across a wide range of organisms. Nevertheless, heterodimeric composition of liver c-AATs from both fish is closer to the mammalian enzyme (Milton et al., 1967a,b; Ishiguro et al., 1991) than those from lower organisms (Chen, 1985; Umemura et al., 1994; Zelada et al., 1996). Thus, the occurrence of subunit shuffling in c-AAT from homodimeric to heterodimeric composition cannot be ruled out. However, between the two fish examined, similar elution profiles of the enzyme on an ion-exchanger,
unchanged pH and temperature optima and comigration of both the subunits on SDS-PAGE appear to indicate that structurally liver c-AATs remain unaltered in the two fish adapted to different modes of life. Nevertheless, purification, percent yields and specific activities of purified preparations (Tables 1 and 2) from C. batrachus were markedly higher than those of L. rohita. The difference becomes more prominent after the ion-exchange chromatography step. Explanations for such differences require more direct evidence. However, the possibility of some alterations in binding affinity of L. rohita c-AAT to the ion-exchanger may not be ruled out. Nonetheless, the final preparation from
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Fig. 7. Double reciprocal plots for determination of KI for AOAA using 2-oxoglutarate as substrate with the purified liver c-AATs from (A) C. batrachus and (B) L. rohita. Each point represents mean value of four observations.
L. rohita was also found to be homogeneous electrophoretically (Fig. 3). The range of Km values of L-alanine and 2oxoglutarate reported in the present study (Table 3) is close to the earlier reports in different vertebrates (Segal et al., 1962; Milton et al., 1967a,b; Patnaik and Kanungo, 1976; Guelbenzu et al., 1991). However, the data presented here exhibit a significantly lower Km for L-alanine in C. batrachus than that in L. rohita. Such a situation at the kinetic level may be correlated with high affinity of c-AAT towards its metabolic substrates in case of a fish adapted to carnivorous and airbreathing mode of life.
AOAA inhibits liver c-AAT competitively (Fig. 6) with respect to its amino acid substrate and uncompetitively (Fig. 7) with respect to the keto acid substrate, in both fish. This indicates that the purified enzymes from both fish have independent binding sites for the two substrates. However, KI values for AOAA with liver c-AAT from C. batrachus were significantly higher, particularly in the presence of L-alanine, than that of the enzyme from L. rohita. Such alterations in KI values of AAT from rat tissues for AOAA have been attributed to differential affinities of the inhibitor for the enzyme, thereby modulating the enzyme activity under different metabolic demands (Patnaik
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and Kanungo, 1976; McKenna et al., 1996). Also, assimilation of L-alanine and 2-oxoglutarate have been found to be extremely low in cases of limited oxygen supply to the organism (Suarez and Mommsen, 1987; Brauner et al., 1999). Therefore, higher affinity for alanine and lower affinity for metabolic inhibitor of the enzyme from C. batrachus seem to be better adapted to catalysis than that of L. rohita. This may be one of the reasons for a significantly higher activity of AAT in liver of C. batrachus than that of L. rohita as observed earlier (Srivastava et al., 1998a). Several studies have reported on the adaptive changes in metabolic enzymes as a function of temperature acclimation in fish (Sidell, 1977; De ¨ Luca et al., 1983; Haschemeyer, 1985; Jurss et al., 1987; Lin and Somero, 1995). However, most information is concerned with the metabolic regulation of the enzyme (Poli et al., 1997; Singh and Singh, 1991; Medda et al., 1995; Mishra and Shukla, 1997). Information on adaptive changes in fish AAT enzyme at the protein level is negligible with a few exceptions (Galichowski et al., 1977; Srivastava et al., 1998b). In the present report, carnivorous, air-breathing C. batrachus and herbivorous, non-air-breathing L. rohita represent two fish with different metabolic needs. Thus, C. batrachus seems to depend more on amino acids than L. rohita for energy production, possibly via malate aspartate shuttle and gluconeogenesis (Thillart and Smit, 1984; Masola et al., 1985; SanchezMuros et al., 1998; Srivastava et al., 1998a; Moraes et al., 2002; Treberg et al., 2003). Therefore, alterations in the kinetics of liver c-AATs from these two fish may be one of the adaptive changes, in favor of higher activity in C. batrachus than that of L. rohita. In conclusion, liver c-AATs purified from C. batrachus and L. rohita show similar physicochemical characteristics. However, differences in kinetic behavior are apparent between the enzymes from the two fish species. Therefore, the possibility of conformational changes in c-AAT may not be ruled out, as the two fish adapt to their different modes of life. Acknowledgments This work was supported by an award of a fellowship from Science and Technology Agency, Japan, and a research fellowship award by The
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¨ Jurss, K., Bittorf, T.H., Volkler, T.H., Wacke, R., 1987. Effects of temperature, food deprivation, and salinity on growth, RNAyDNA ratio and certain enzyme activities in rainbow trout (Salmo gairdneri). Comp. Biochem. Physiol. B 87, 241–253. Keilin, D., Hartree, E.F., 1938. Preparation of calcium phosphate gel. Methods Enzymol. 1, 98. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 227, 680–685. Lin, J., Somero, G.N., 1995. Temperature dependent changes in expression of thermostable and thermolabile isoenzymes of cytosolic malate dehydrogenase in eurythermal goby fish (Gillichtys mirabilis). Physiol. Zool. 68, 114–128. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. Malbrouck, C., Trausch, G., Devos, P., Kestemont, P., 2003. Hepatic accumulation and effects of microcystin-LR on juvenile goldfish Carassius auratus L. Comp. Biochem. Physiol. B 135, 39–48. Masola, B., Peters, T.J., Evered, D.F., 1985. Transamination pathways influencing L-glutamine and L-glutamate oxidation by rat enterocyte mitochondria and the subcellular localization of L-alanine aminotransferase and L-aspartate aminotransferase. Biochim. Biophys. Acta 843, 137–143. Matsuzawa, T., Segal, H.L., 1968. Rat liver alanine aminotransferase-crystallization, composition and role of sulfhydryl groups. J. Biol. Chem. 243, 5929–5934. McKenna, M.C., Tildon, J.T., Stevenson, J.H., Huang, X., 1996. New insights into the compartmentation of glutamate and glutamine in cultured rat brain astrocytes. Dev. Neurosci. 18, 380–390. Medda, C., Bhatacharya, B., Sarkar, S.K., Ganguli, S., Basu, T.K., 1995. Effect of rotenone on activity of some enzymes and their recovery in fresh water carp fingerlings of L. rohita. J. Environ. Biol. 16, 55–60. Milton, H., Saeir, J., Jenkins, W.T., 1967. Alanine aminotransferase: purification and properties. J. Biol. Chem. 242, 91–100. Milton, H., Saier, J., Jenkins, W.T., 1967. Alanine aminotransferase, the basis for substrate specificity. J. Biol. Chem. 242, 101–108. Mishra, R.K., Shukla, S.P., 1997. Impact of endosulfan on cytoplasmic and mitochondrial liver malate dehydrogenase from the fresh water catfish (Clarias batrachus). Comp. Biochem. Physiol. C 117, 7–18. Mommsen, T.P., Osachoff, H.L., Elliott, M.E., 2003. Metabolic zonation in teleost gastrointestinal tract effects of fasting and cortisol in tilapia. J. Comp. Physiol. B 173, 409–417. Moraes, G., Avilez, I.M., Altran, A.E., Barbosa, C.C., 2002. Biochemical and hematological responses of the banded knife fish Gymnotus carapo (Linnaeus, 1758) exposed to environmental hypoxia. Braz. J. Biol. 62, 633–640. Patnaik, S.K., Kanungo, M.S., 1976. Soluble alanine aminotransferase of the liver of rats of various ages: induction, characterization and changes in patterns. Ind. J. Biochem. Biophys. 13, 117–124. Poli, A., Notori, S., Virgili, M., Fabbri, E., Lucchi, R., 1997. Neurochemical changes in cerebellum of gold fish exposed to various temperatures. Neurochem. Res. 22, 141–149.
Renaud, J.M., Moon, T.W., 1980. Starvation and metabolism of hepatocytes isolated from the American eel Anguilla rostrata. J. Comp. Physiol. 135, 127–137. Rodriguez, I.E., Bartolome, J., Lopeg-Alcorocho, J.M., Contonat, T., Oliva, H., Carreno, V., 1997. Activation of liver diseases in healthy hepatitis B surface antigen carriers during its treatment. J. Med. Virol. 53, 76–80. Samsonova, M.V., Min’kova, N.O., Lapteva, T.I., Mikodina, E.V., 2003. Filippovich LuB. Aspartate- and alanine-aminotransferase in early development of keta. Ontogenez 34, 19–23. Sanchez-Muros, M.J., Garcia-Rejon, L., Garcia-Salguero, L., de la Higuera, M., Lupianez, J.A., 1998. Long-term nutritional effects on the primary liver and kidney metabolism in rainbow trout. Adaptive response to starvation and a high-protein, carbohydrate-free diet on glutamate dehydrogenase and alanine aminotransferase kinetics. Int. J. Biochem. Cell Biol. 30, 55–63. Segal, H.L., Beattle, D.S., Hopper, S., 1962. Purification and properties of liver glutamic-alanine transaminase from normal and corticoid treated rats. J. Biol. Chem. 237, 1914–1920. Segal, H.L., Matsuzawa, T., 1970. Alanine aminotransferase from rat liver. Methods Enzymol. 17, 153–159. Sidell, B.D., 1977. Turnover of cytochrome C in skeletal muscle of green sunfish (Lepomis cyanellus) during thermal acclimation. J. Exp. Zool. 199, 233–250. Singh, R.A., Singh, S.N., 1991. Kinetic characterization and regulation of purified glutamine synthetase from brain of C. batrachus. Arch. Int. Physiol. Biochim. 98, 95–101. Singh, R.A., Singh, S.N., 1992. In vitro effects of divalent metal ions and metabolic modulators of purified glutamine synthetase from brain of teleostean fish. Arch. Int. Physiol. Biochim. Biophys. 100, 203–205. Srivastava, A.S., Oohara, I., Suzuki, T., Singh, S.N., 1998. Activity and expression of glutamate oxaloacetate transaminase during the reproductive cycle of fresh water fish Labeo rohita. Fish. Sci. 64, 621–626. Srivastava, A.S., Trigun, S.K., Singh, S.N., 1998. Activity of cytosolic and mitochondrial alanine aminotransferase during pre-spawning phase of air-breathing and non-air-breathing fresh water fish. J. Sci. Res. 47, 163–171. Srivastava, A.S., Oohara, I., Suzuki, T., Singh, S.N., 1999. Activity and expression of aspartate aminotransferase during the reproductive cycle of a fresh water fish, Clarias batrachus. Fish Physiol. Biochem. 20, 243–250. Suarez, R.K., Mommsen, T.P., 1987. Gluconeogenesis in teleost fishes. Can. J. Zool. 65, 1869–1882. Sunny, F., Jacob, A., Oommen, O.V., 2002. Sex steroids regulate intermediary metabolism in Oreochromis mossambicus. Endocr. Res. 28, 175–188. Thillart, G.V.D., Smit, H., 1984. Carbohydrate metabolism of goldfish (Carassius auratus L.) effect of long term hypoxiaacclimation on enzyme pattern of red muscle, white muscle and liver. J. Comp. Physiol. B 154, 477–486. Treberg, J.R., Lewis, J.M., Driedzic, W.R., 2003. Comparison of liver enzyme in osmerid fishes: key differences between a glycerol accumulating species, rainbow smelt (Osmerus mordax), and a species that does not accumulate glycerol, capelin (Mallotus villosus). Comp. Biochem. Physiol. B 132, 433–438.
A.S. Srivastava et al. / Comparative Biochemistry and Physiology Part B 137 (2004) 197–207 Umemura, I., Yanagiya, K., Komatsubara, S., Sato, T., Tosa, T., 1992. D-alanine production from DL-alanine by Candida maltosa with asymmetric degrading activity. Appl. Microbiol. Biotechnol. 36, 722–726. Umemura, I., Yanagiya, K., Komatsubara, S., Sato, T., Tosa, T., 1994. Purification and some properties of alanine aminotransferase from Candida maltosa. Biosci. Biotechnol. Biochem. 58, 283–287. Walton, M.J., Cowey, C.B., 1982. Aspects of intermediary metabolism in salmonid fish. Comp. Biochem. Physiol. B 72, 59–79.
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Zelada, C., Montemartini, M., Cazzulo, J.J., Nowicki, C., 1996. Purification and partial structural and kinetic characterization of an alanine aminotransferase from epimastigotes of Trypanosoma cruzi. Mol. Biochem. Parasitol. 79, 225–228. Zinkel, J.G., Bush, R.M., Cormelius, C.E., Freedland, R.A., 1971. Comparative studies on plasma and tissue sorbitol, glutamic acid, lactic acid and hydroxybutyric hydrogenase and transaminase activities in the dog. Res. Vet. Sci. 12, 211–214.
Purification and properties of cytosolic alanine aminotransferase from the liver of two freshwater fish, Clarias batrachus and Labeo rohita Anand S. Srivastavaa, Ichiro Ooharab, Tohru Suzukic, Steve Shenoudaa, Surender N. Singhd, Dharam P. Chauhana, Ewa Carriera,* a School of Medicine, University of California San Diego, La Jolla, CA 92093, USA National Research Institute of Fisheries Science, Fukuura 2-12-4 Kanazawa, Yokohama 236-8648, Japan c Metabolism Section, Fish Nutrition Division, National Research Institute of Aquaculture, Nansei Watarei, Mie, 516-01, Japan d Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi 221 005, India b
Received 25 July 2003; received in revised form 8 November 2003; accepted 10 November 2003
Abstract Cytosolic alanine aminotransferase (c-AAT) was purified up to 203- and 120-fold, from the liver of two freshwater teleosts Clarias batrachus (air-breathing, carnivorous) and Labeo rohita (water-breathing, herbivorous), respectively. The enzyme from both fish showed similar elution profiles on a DEAE-Sephacel ion exchange column. SDS-PAGE of purified enzymes revealed two subunits of 54 and 56 kDa, in both fish. The apparent Km values for L-alanine were 18.5"0.48 and 23.55"0.60 mM, whereas for 2-oxoglutarate the Km values were observed to be 0.29"0.023 and 0.33"0.028 mM for the enzyme from C. batrachus and L. rohita, respectively. With L-alanine as substrate, aminooxyacetic acid was found to act as a competitive inhibitor with KI values of 6.4=10y4 and 3.4=10y4 mM with c-AAT of C. batrachus and L. rohita, respectively. However, when 2-oxoglutarate was used as substrate, aminooxyacetic acid showed uncompetitive inhibition with similar KI values for purified c-AAT from both fish. Temperature and pH profiles of the enzyme did not show any marked differences between the two fish examined. These results suggest that liver c-AAT, isolated from these two fish species adapted to different modes of life, remain unaltered structurally. However, at the kinetic level, liver c-AAT from C. batrachus exhibits significantly higher affinity for the substrate Lalanine and decreased affinity for its metabolic inhibitor, in comparison to that of the enzyme purified from L. rohita. Such functional changes seem to be of physiological significance and also provide preliminary evidence for subtle changes in the enzyme as a mark of metabolic adaptation in the fish to different physiological demands. 䊚 2003 Elsevier Inc. All rights reserved. Keywords: Alanine aminotransferase; Enzyme; Fish; GPT
1. Introduction Fish show remarkable capacity to utilize proteins and amino acids for both, energy production and biosynthetic processes (Renaud and Moon, 1980; *Corresponding author. Tel.: q1-858-822-1050; fax: q1858-822-0835. E-mail address: [email protected] (E. Carrier).
Suarez and Mommsen, 1987; Foster and Moon, 1991; Treberg et al., 2003; Mommsen et al., 2003). Obviously transamination reactions of amino acids are one of the critical steps in setting the pace of several metabolic events in fish. Alanine aminotransferase (AAT; EC: 2.6.1.2.), a pyridoxal-phosphate-dependent enzyme, has been observed to be one of the sensitive and key enzymes in this
1096-4959/04/$ - see front matter 䊚 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2003.11.006
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respect (Walton and Cowey, 1982). The enzyme, compartmentalized in cytosol (c-AAT) and mitochondria (m-AAT), is responsible for reversible transamination between alanine and 2-oxoglutarate to form pyruvate and glutamate. As a key enzyme that provides the substrate for the malate aspartate shuttle (Masola et al., 1985), this reaction becomes instrumental in transport of crucial metabolites across the mitochondrial membrane and thereby channeling alanine into general metabolic pathways. The profile of this enzyme has been shown to vary with age, sex and in a tissue-specific manner in several animals studied (Zinkel et al., 1971; Alexin and Papoutsoglou, 1986; Abraham and Rina, 1991). Extensive information on AAT is available from mammalian models. Crystallized cAAT from rat has been reported to contain two closely migrating sub-bands on PAGE (Matsuzawa and Segal, 1968), similar to those of the noncrystallized homogeneous enzyme (Gatehouse et al., 1967). The molecular mass of c-AAT was calculated to be 114 kDa from rat liver (Segal et al., 1962) and 90 and 115 kDa from pig heart (Milton et al., 1967a,b). Ishiguro et al. (1991) have worked out the complete amino acid sequence of the 55-kDa subunit of c-AAT from rat liver. AAT isolated and characterized from Drosophila nigromelanica was also shown to be a homodimer of 113 kDa (Chen, 1985). Extensive studies have been done on the metabolic importance of this enzyme in a number of mammals. In addition, AAT profiles have been suggested as one of the markers of a variety of viral and other infections in rat and human (Agarawal et al., 1996; Cahen et al., 1996; Rodriguez et al., 1997). In fish, however, available information is restricted to the alterations in AAT activities as a function of environmental factors and dietary compositions (Alexin and Papoutsoglou, 1986; ¨ Jurss et al., 1987; Foster and Moon, 1991; Sunny et al., 2002; Treberg et al., 2003; Malbrouck et al., 2003; Samsonova et al., 2003). We have demonstrated that different food habits and respiratory patterns bring about significant adaptive changes in the activity of c-AAT and aspartate aminotransferase (AspT) in different tissues of Clarias batrachus and Labeo rohita (Srivastava et al., 1998a,b, 1999). To study the mechanism of such changes in AAT, we now intend to address the question, ‘How does AAT adapt at the protein level as a function of different metabolic demands?’ Also,
though AAT has been purified from mammals, Drosophila (Chen, 1985), Candida maltosa (Umemura et al., 1992), Trypanosoma (Zelada et al., 1996), etc. studies on purification and macromolecular properties of the enzyme have not been a focus in freshwater fish. In this paper we report on the purification, characterization and structure– function relationship of c-AAT from the liver of two economically important freshwater fish of south-eastern Asia, C. batrachus and L. rohita. C. batrachus is a carnivorous air-breathing catfish, with suprabranchial arborescent accessory airbreathing organ that functions much like a lung. The cyprinid L. rohita is an herbivorous waterbreathing fish. 2. Materials and methods 2.1. Animals Female freshwater teleosts C. batrachus and L. rohita were collected from River Ganga in Varanasi during June and July (spawning phase of fish). C. batrachus, weighing 70–75 g and 18–20 cm length, and L. rohita, 150 g and 20–22 cm, were killed at approximately 10.00–11.00 h to avoid the effect of circadian rhythmicity for all experiments. 2.2. Chemicals Analytical grade chemicals were used. Biochemicals used were purchased from Sigma Chemical Co., St. Louis, MO. DEAE-Sephacel and LMW calibration kits were obtained from Pharmacia Fine Chemicals. Calcium phosphate gel was prepared in our laboratory according to the method of Keilin and Hartree (1938). 2.3. Assay of AAT The activity of c-AAT was assayed spectrophotometrically following the method of Segal and Matsuzawa (1970). The reaction mixture consisted of potassium phosphate buffer, 0.1 M (pH 7.4); L-alanine, 0.2 M; lactate dehydrogenase, 50 unitsy ml; NADH, 5.6 mM; 2-oxoglutarate, 0.25 M. Rate of conversion of alanine to pyruvate was monitored by coupling the reaction with the formation of lactate from pyruvate in the presence of NADH at 25 8C. The reaction was started by addition of 2oxoglutarate, and the decrease in optical density
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was recorded at 340 nm. A unit of enzyme activity was defined as the amount of enzyme that converts 1 mmol of NADH per minute at 25 8C. 2.4. Purification of c-AAT Cytosolic alanine aminotransferase was purified following the methods of Gatehouse et al. (1967) with slight modifications. All steps were carried out at 4"2 8C unless otherwise indicated. Immediately after killing the fish the liver was removed and washed in ice-cold 0.69% NaCl solution. Tissue was homogenized (10% wyv) in ice-cold 0.25 M sucrose solution containing 1 mM EDTA. Homogenate was centrifuged at 15 000=g for 30 min. Supernatant was subjected to heat treatment at 55 8C followed by acid precipitation at pH 5.5 by 1 M acetic acid. Supernatant collected after centrifugation at 10 000=g was used for ammonium sulfate fractionation. The pellet collected after 45% ammonium sulfate precipitation was dissolved in a minimum volume of 10 mM potassium phosphate buffer, pH 5.7, containing 1 mM dithiothreitol (DTT). Sample was dialyzed overnight against the same buffer. Dialysate was cleared at 40 000=g for 3 h. The clear fraction was applied to a 15=2 cm DEAE-Sephacel column equilibrated with 10 mM potassium phosphate buffer, pH 5.7, containing 1 mM DTT. The column was developed in the equilibrating buffer at a flow rate of 0.9 mlymin. The enzyme was eluted by the application of the same buffer containing 0.025 mM Na2SO4. Multiple fractions were collected and assayed for enzyme activity. Fractions with more than 40% enzyme activity were pooled and used for calcium phosphate gel treatment. A calcium phosphate gel suspension was mixed with the total volume of pooled fractions in the ratio of 20 mgy ml. After 5 min of gentle mixing, the gel was removed by centrifugation at 10 000=g. This process was repeated three times using fresh gel suspension each time. The treated fraction was concentrated by 50% ammonium sulfate precipitation. The pellet obtained was dissolved in 2.0ml 0.1 M potassium phosphate buffer, pH 7.5, containing 0.1 M L-proline and 1 mM DTT. The solution was chilled and 6.0-ml cold acetone maintained at y20 8C was added slowly and the tube was centrifuged immediately at 0 8C. The pellet was then dissolved in 2.0-ml 0.1 M potassium phosphate buffer, pH 7.5, containing 0.1 M Lproline and 1 mM DTT and centrifuged. After
199
55% ammonium sulfate precipitation, the pellet was dissolved in a minimum volume of 0.1 M potassium phosphate buffer, pH 7.5, containing 1 mM DTT and EDTA. The solution was centrifuged again and the cleared suspension was stored at 4 8C. This was treated as the purified enzyme preparation. 2.5. Analytical methods The protein concentrations of the samples were determined by the Folin method (Lowry et al., 1951). For molecular weight determination of liver c-AAT, 20-mg desalted protein samples along with MW markers were subjected to 10% SDS-PAGE following the method of Laemmli (1970). After electrophoresis, gels were stained in 0.1% Coomassie brilliant blue R-250 and photographed. 2.6. In vitro studies The Km value for L-alanine of purified liver cAAT was determined by monitoring the enzyme activity at different concentrations of L-alanine (10–100 mM). 2-Oxoglutarate was kept constant at a saturating concentration (0.3 M) in these assays. The Km value for 2-oxoglutarate was determined by measuring the enzyme activity at varying concentration of the substrate, keeping L-alanine at its saturation level (0.25 M). Lineweaver–Burk plots of the data were used to determine the Km values. The effect of aminooxyacetate on the forward reaction was observed separately with respect to both substrates. The enzyme samples were assayed as described earlier, at different concentrations of aminooxyacetate (AOAA). KI values were calculated from a double reciprocal plot of the data. To study the effect of temperature, suitably diluted enzymes were incubated separately at different temperatures (15–70 8C) for 10 min in the standard assay mixture, and remaining activity was determined at 25 8C as described in the text. The effect of pH was monitored by incubating the enzyme samples separately in assay mixtures consisting of phosphate buffers over a pH range of 5.5–9.5 for 10 min at 37 8C followed by enzyme assay at 25 8C. 2.7. Statistical analysis Averages and standard deviations were obtained from four values each time. Student’s t-test was used to test significances.
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Fig. 1. Elution profile of liver c-AAT of C. batrachus on DEAE-Sephacel column. Ammonium sulfate fractionated dialyzed sample was applied on the column and elution was made as described in the text.
Fig. 2. Elution profile of liver c-AAT of L. rohita on DEAESephacel column. Ammonium sulfate fractionated dialyzed sample was applied on the column and elution was made as described in the text.
3. Results with a 38% yield from C. batrachus. The enzyme was purified 120-fold from the liver of L. rohita with a 27% yield. Nevertheless, both the preparations were observed to be nearly homogenous on SDS-PAGE (Fig. 3). SDS-PAGE results also exhibit that the final preparations from both the fish contain two closely migrating c-AATs (A and B) of 54 and 56 kDa,
Figs. 1 and 2 show that the enzyme c-AAT from both C. batrachus and L. rohita is eluted from a DEAE-Sephacel column following a similar elution pattern. Summary of stepwise purification protocol is presented in Tables 1 and 2. Combining two batch separations after ion-exchange chromatography, the liver c-AAT was purified 202-fold Table 1 Summary of purification of liver c-AAT of C. batrachus Steps of purification
Volume (ml)
Total units
Specific activity
Fold of purification
% yield
(1) (2) (3) (4) (5) (6)
128 118 20 10 12 6
472 427 303 250 223 180
0.44 0.47 3.00 39.7 77.5 89.5
1.7 6.7 89.8 175 202
100 90 64 53 47 38
Cytosolic fraction (15 000=g) Heat extraction (NH4)2SO4 precipitation DEAE-Sephacel column Ca3(PO4)2 Gel Acetone precipitation
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Table 2 Summary of purification of liver c-AAT of L. rohita Steps of purification
Volume (ml)
Total units
Specific activity
Fold of purification
% yield
(1) (2) (3) (4) (5) (6)
125 116 18 10 12 5.5
683 507 413 309 259 82
0.41 0.53 1.8 26.2 44.4 50.0
– 1.3 4.2 59.5 107 120
100 74 60 45 38 27
Cytosolic fraction (15 000=g) Heat extraction (NH4)2SO4 precipitation DEAE-Sephacel column Ca3(PO4)2 Gel column Acetone precipitation
as determined on semilogarithmic plot (plot not shown) prepared against standard molecular weight marker proteins. No differences in MW of liver c-AATs between the two species were noted. 3.1. Temperature and pH studies Fig. 4 presents the percent activity of purified c-AATs incubated and assayed at different temperatures. The temperature maximum is observed to be 37 8C for liver c-AAT from both fish. Temperature profiles at lower temperatures appear to be unaltered with the enzyme from both fish, whereas higher temperature ranges show significant inactivation in the enzyme activity of C. batrachus. Profiles of liver c-AAT from both fish exhibit significant differences at lower pH ranges with pH optima of 7.5 (Fig. 5).
Fig. 3. Ten percentage SDS-PAGE of liver c-AAT fractions at various stages of purification. (1) Twenty microgram protein was loaded from crude homogenate, (2) ammonium sulfate fraction, (3) DEAE-Sephacel elute and (4) purified preparation. M, marker protein; C, C. batrachus; L, L. rohita. Final preparations from both the fish contain two closely migrating c-AATs (A and B) of 54 and 56 kDa, as determined on semilogarithmic plot (plot not shown) prepared against standard molecular weight marker proteins.
3.2. Kinetics Table 3 indicates that apparent kinetic constants Km for L-alanine are calculated to be 18.5"0.48 and 23.5"0.6 mM and for 2-oxoglutarate values are 0.29"0.023 and 0.33"0.028 mM with liver c-AAT purified from C. batrachus and L. rohita, respectively. In the presence of L-alanine, AOAA acts as competitive inhibitor with KI values of 6.4=10y4 and 3.8=10y4 mM for liver c-AAT from C. batrachus and L. rohita, respectively (Fig. 6). AOAA behaves as an uncompetitive inhibitor
Fig. 4. Effect of temperature on purified c-AATs from the liver of C. batrachus (q) and L. rohita (D). The activity observed at 37 8C was taken as 100% and mean value of four observations has been plotted on each point. Each value is presented with the form of mean"S.D. ***P-0.001; *P-0.05.
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Fig. 5. Effect of pH on purified c-AATs from the liver of C. batrachus (q) and L. rohita (D). The activity observed at pH 7.5 was taken as 100% and mean value of four observations has been plotted on each point. Each value is presented with the form of mean"S.D. ***P-0.001; *P-0.01; **P-0.05.
of the enzyme in the presence of 2-oxoglutarate with similar KI values (4.8=10y4 and 4.3=10y4 mM; Fig. 7). 4. Discussion Alterations in key metabolic enzymes are instrumental in mediating metabolic adaptive changes in cells under the influence of external and internal factors. Enzymes have tremendous capacity to adjust at the structural level in this respect. Therefore, studies on structure–function relationship of key enzymes are of special interest. Particularly in fish, AAT has been found to show significant alterations under the influence of environmental factors and dietary composition. These changes in AAT activity may be due to alterations in the concentration of AAT in the tissues (Alexin and ¨ Papoutsoglou, 1986; Jurss et al., 1987; Foster and Moon, 1991). We have observed that different tissues of air-breathing carnivorous fish show significant increases in its activity compared to nonair-breathing herbivorous fish (Srivastava et al., 1998a). To understand the mechanism of such changes in c-AAT at the protein level, the enzyme was purified from the liver of C. batrachus and L.
rohita. Since literature is not available on the isolation of AATs from freshwater fish, we followed the method reported in other animal models with modifications realized during standardization of the procedure. To avoid denaturation of the enzymes during the entire process, appropriate concentrations of DTT and EDTA were included in all solutions. The pH was monitored and maintained continuously during the ammonium sulfate precipitation; L-proline was added to protect the prosthetic group of the enzyme as reported earlier (Segal and Matsuzawa, 1970). Summary of the purification indicates a twostep ion-exchange chromatography and acetone fractionation results in isolation of liver c-AAT to electrophoretic homogeneity from both C. batrachus and L. rohita. Similar purification results have also been reported from other animals (Segal and Matsuzawa, 1970; Patnaik and Kanungo, 1976; Guelbenzu et al., 1991; Umemura et al., 1992). Separation of two c-AAT peaks on ionexchange chromatography, one in unbound and another in bound fractions, also corroborates earlier reports in other animals (Harding et al., 1961; Chen and Giblett, 1971; Patnaik and Kanungo, 1976; Umemura et al., 1992, 1994). SDS-PAGE of the final preparation from the second peak of the liver c-AATs revealed two closely migrating protein bands of 54 and 56 kDa. If it is assumed that the native protein is a heterodimer of 110 kDa in the case of both fish, values are close to cAATs from pig cardiac muscle, rat liver (Milton et al., 1967a,b), human liver (Ishiguro et al., 1991) and Chlaymydomonas reinharditii (Guelbenzu et al., 1991). From our laboratory, another enzyme, glutamine synthetase from the brain of C. batrachus, has also been reported to be structurally close to the enzyme from mammals (Singh and Singh, 1992). Temperature and pH profiles of purified liver c-AATs from both fish are almost the same. The pH optima corroborate the earlier Table 3 Apparent Km values of purified liver c-AATs of C. batrachus and L. rohita with L-alanine and 2-oxoglutarate Substrate Fish
L-alanine
Km (mM)
2-oxoglutarate Km (mM)
C. batrachus L. rohita
18.50"0.48 23.55"0.60***
0.29"0.023 0.33"0.028
*** P-0.001 represents the comparison between two species. Mean"S.D. (ns4).
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Fig. 6. Double reciprocal plots for determination of KI for AOAA using L-alanine as substrate with the purified liver c-AATs from (A) C. batrachus and (B) L. rohita. Each point in the plot represents mean value of four observations.
reports from other vertebrates (Segal et al., 1962; Matsuzawa and Segal, 1968; Galichowski et al., 1977). Thus, it appears like glutamine synthetase (Singh and Singh, 1992), functionally c-AAT has not undergone much evolutionary change across a wide range of organisms. Nevertheless, heterodimeric composition of liver c-AATs from both fish is closer to the mammalian enzyme (Milton et al., 1967a,b; Ishiguro et al., 1991) than those from lower organisms (Chen, 1985; Umemura et al., 1994; Zelada et al., 1996). Thus, the occurrence of subunit shuffling in c-AAT from homodimeric to heterodimeric composition cannot be ruled out. However, between the two fish examined, similar elution profiles of the enzyme on an ion-exchanger,
unchanged pH and temperature optima and comigration of both the subunits on SDS-PAGE appear to indicate that structurally liver c-AATs remain unaltered in the two fish adapted to different modes of life. Nevertheless, purification, percent yields and specific activities of purified preparations (Tables 1 and 2) from C. batrachus were markedly higher than those of L. rohita. The difference becomes more prominent after the ion-exchange chromatography step. Explanations for such differences require more direct evidence. However, the possibility of some alterations in binding affinity of L. rohita c-AAT to the ion-exchanger may not be ruled out. Nonetheless, the final preparation from
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Fig. 7. Double reciprocal plots for determination of KI for AOAA using 2-oxoglutarate as substrate with the purified liver c-AATs from (A) C. batrachus and (B) L. rohita. Each point represents mean value of four observations.
L. rohita was also found to be homogeneous electrophoretically (Fig. 3). The range of Km values of L-alanine and 2oxoglutarate reported in the present study (Table 3) is close to the earlier reports in different vertebrates (Segal et al., 1962; Milton et al., 1967a,b; Patnaik and Kanungo, 1976; Guelbenzu et al., 1991). However, the data presented here exhibit a significantly lower Km for L-alanine in C. batrachus than that in L. rohita. Such a situation at the kinetic level may be correlated with high affinity of c-AAT towards its metabolic substrates in case of a fish adapted to carnivorous and airbreathing mode of life.
AOAA inhibits liver c-AAT competitively (Fig. 6) with respect to its amino acid substrate and uncompetitively (Fig. 7) with respect to the keto acid substrate, in both fish. This indicates that the purified enzymes from both fish have independent binding sites for the two substrates. However, KI values for AOAA with liver c-AAT from C. batrachus were significantly higher, particularly in the presence of L-alanine, than that of the enzyme from L. rohita. Such alterations in KI values of AAT from rat tissues for AOAA have been attributed to differential affinities of the inhibitor for the enzyme, thereby modulating the enzyme activity under different metabolic demands (Patnaik
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and Kanungo, 1976; McKenna et al., 1996). Also, assimilation of L-alanine and 2-oxoglutarate have been found to be extremely low in cases of limited oxygen supply to the organism (Suarez and Mommsen, 1987; Brauner et al., 1999). Therefore, higher affinity for alanine and lower affinity for metabolic inhibitor of the enzyme from C. batrachus seem to be better adapted to catalysis than that of L. rohita. This may be one of the reasons for a significantly higher activity of AAT in liver of C. batrachus than that of L. rohita as observed earlier (Srivastava et al., 1998a). Several studies have reported on the adaptive changes in metabolic enzymes as a function of temperature acclimation in fish (Sidell, 1977; De ¨ Luca et al., 1983; Haschemeyer, 1985; Jurss et al., 1987; Lin and Somero, 1995). However, most information is concerned with the metabolic regulation of the enzyme (Poli et al., 1997; Singh and Singh, 1991; Medda et al., 1995; Mishra and Shukla, 1997). Information on adaptive changes in fish AAT enzyme at the protein level is negligible with a few exceptions (Galichowski et al., 1977; Srivastava et al., 1998b). In the present report, carnivorous, air-breathing C. batrachus and herbivorous, non-air-breathing L. rohita represent two fish with different metabolic needs. Thus, C. batrachus seems to depend more on amino acids than L. rohita for energy production, possibly via malate aspartate shuttle and gluconeogenesis (Thillart and Smit, 1984; Masola et al., 1985; SanchezMuros et al., 1998; Srivastava et al., 1998a; Moraes et al., 2002; Treberg et al., 2003). Therefore, alterations in the kinetics of liver c-AATs from these two fish may be one of the adaptive changes, in favor of higher activity in C. batrachus than that of L. rohita. In conclusion, liver c-AATs purified from C. batrachus and L. rohita show similar physicochemical characteristics. However, differences in kinetic behavior are apparent between the enzymes from the two fish species. Therefore, the possibility of conformational changes in c-AAT may not be ruled out, as the two fish adapt to their different modes of life. Acknowledgments This work was supported by an award of a fellowship from Science and Technology Agency, Japan, and a research fellowship award by The
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