Purification and kinetic characteristics of strombine dehydrogenase from the foot muscle of the hard clam (Meretrix lusoria)

Comparative Biochemistry and Physiology, Part B 158 (2011) 38–45

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Comparative Biochemistry and Physiology, Part B j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p b

Purification and kinetic characteristics of strombine dehydrogenase from the foot muscle of the hard clam (Meretrix lusoria) An-Chin Lee ⁎, Kuen-Tsung Lee, Li-Ying Pan Department of Aquatic Biosciences, College of Life Science, National Chiayi University, Chiayi 600, Taiwan

a r t i c l e

i n f o

Article history: Received 25 July 2010 Received in revised form 7 September 2010 Accepted 8 September 2010 Available online 16 September 2010 Keywords: Characteristics Hard clam Purification Strombine dehydrogenase

a b s t r a c t Strombine dehydrogenase (SDH, EC 1.5.1.22) from the foot of the hard clam Meretrix lusoria was purified over 470-fold to apparent homogeneity. It has a monomeric structure with a relative molecular mass of 46,000. Two isoenzymes were identified with isoelectric points of 6.83 and 6.88. SDH is heat labile, and has pH and temperature optima of 7.4–7.6 and 45–46 °C, respectively. L-Alanine, glycine, and pyruvate are the preferred substrates. L-Serine is the third preferred amino acid. Iminodiacetate with the lowest Ki of SDH at both pH 6.5 and 7.5 was the strongest inhibitor among succinate, acetate, iminodiacetate, oxaloacetate, and L-/D-lactate. The inhibitory activities of succinate at pH 6.5, and iminodiacetate and oxaloacetate at pH 7.5 on the SDH were higher. These inhibitors are either competitive or mixed-competitive inhibitors. Half of the enzymatic activity of SDH was inhibited by 0.2 mM Fe3+ and 0.6 mM Zn2+. © 2010 Elsevier Inc. All rights reserved.

1. Introduction Hard clams (Meretrix lusoria) are very important economic bivalves and their production among the malacofauna in Taiwan was the first in 2008 (Fisheries Agency, 2008). They are benthic marine organisms and naturally inhabit estuaries and intertidal regions. Therefore, these organisms have developed anaerobiosis to cope with exposure stress, enabling them to survive during low tide (Lee et al., 2008). However, most hard clams sold in the market are cultured in ponds in the southwestern part of Taiwan. Usually, powdered fish meal and fermented organic matter are used to directly feed the clams and enrich natural production. A reduced layer in the sediments is commonly formed due to the residues of added organic matter and feces of the hard clam (Hon, 1988). The reduced layer consumes a lot of dissolved oxygen (DO) and results in low levels of DO in the bottom layer of pond water under stagnant conditions. Therefore, hard clams, even when not subjected to aerial exposure, are also likely to experience environmental hypoxia in aged ponds. Anaerobiosis is divided into two categories, environmental and functional anaerobiosis. The glucose-succinate and aspartate-succinate pathways are widely recognized to provide chemical energy to organisms under environmental anoxia (de Zwaan, 1983; Grieshaber et al., 1994). However, the lactate and opine pathways provide organisms with instantaneous anaerobic energy production under functional anoxia (Livingstone, 1983; Livingstone, 1991). The lactate ⁎ Corresponding author. Department of Aquatic Biosciences, College of Life Sciences, National Chiayi University, 300 University Road, Chiayi 600, Taiwan. Tel.: + 886 5 2717849; fax: + 886 5 2717847. E-mail address: [email protected] (A.-C. Lee). 1096-4959/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2010.09.003

pathway characterized by a single substrate (pyruvate) and end product (lactate) is the best known and most researched anaerobic pathway. The energetic efficiency of the lactate pathway is low compared to catabolism of glucose in the tricarboxylic acid (TCA) cycle, but the rate of energy production is relatively much higher than aerobic catabolism (Livingstone, 1983). The opine pathway is similar to the lactate pathway. In addition to NADH and NAD+, opine dehydrogenase requires two substrates (pyruvate and amino acids) and produces only one product (opine). These enzymes generally have broad amino acid specificity and similar catalytic properties (Gäde and Grieshaber, 1986). They are normally monomeric proteins and widely distributed in marine invertebrates (de Zwaan and Zurburg, 1981; Fields and Hochachka, 1981; Gäde and Carlsson, 1984; Kanno et al., 1996, 1999; Storey, 1983). The classification of opine dehydrogenases is completely dependent on one of the two substrates, amino acids. Thus far, six different opines and corresponding opine dehydrogenases have been reported. These enzymes are strombine dehydrogenase (SDH; with glycine as a substrate), alanopine dehydrogenase (ADH; EC 1.5.1.17; with alanine as a substrate), octopine dehydrogenase (ODH; EC 1.5.1.11; with arginine as a substrate), tauropine dehydrogenase (TDH; EC 1.5.1.23; with taurine as a substrate), β-alanopine dehydrogenase (BADH; EC 1.5.1.26; with β-alanine as a substrate), and lysopine dehydrogenase (LYDH; EC 1.5.1.16; with lysine as a substrate) (Kanno et al., 2005). SDH and TDH are recognized as enzymes in the most ancient opine pathways among these opine pathways, while the octopine pathway is the most advanced (Livingstone, 1991). SDH (originally called ADH) was first found in the adductor muscle of the oyster, Crassostrea gigas (Fields et al., 1980). The enzyme was renamed SDH according to the definition by Dando et al. (1981). They

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defined ADH as having a higher specificity for alanine than for glycine. By contrast, SDH utilizes glycine as its preferred amino acid substrate but also readily reacts with alanine. SDH was also found in the adductor muscle of other bivalves (Dando et al., 1981; de Zwaan and Zurburg, 1981; Nicchitta and Ellington, 1984), as well as in polychaetes (Storey, 1983) and sponges (Barrett and Butterworth, 1981; Plese et al., 2009). The enzyme was purified, and its kinetics were studied in the sea mouse Aphrodite aculeata (Storey, 1983), from the foot muscle of the cherrystone clam Mercenaria mercenaria (Linn.) (Fields and Storey, 1987) and from the demosponge Suberites domuncula (Plese et al., 2009). A study of the distribution of opine dehydrogenases in tissues of hard clams showed that ADH, ODH, and SDH are the predominant opine dehydrogenases in the foot and adductor of the hard clam under normoxia (Lee and Lee, in press). After anoxic exposure, an increase in the activity of SDH was the highest in the foot of the hard clam. Therefore, SDH may play an important role in the physiological functioning of hard clams under anaerobiosis. In order to better assess its physiological role in the metabolism, SDH was first isolated from the foot of hard clams. Studied on molecular characteristics, substrate specificity, and kinetic properties of SDH were taken. This paper is also concerned with the inhibitory effect of metabolites, storage conditions, and the effects of ions on the enzymatic activities of SDH. 2. Material and methods 2.1. Materials Hard clams (15.6 ± 0.6 g) were purchased from clam farms in the Taishi area (Yunlin County, southwestern Taiwan) and starved for 2 days in 2000-L tanks containing 1300 L of 20‰ artificial seawater (ASW) prepared by dissolving 1:50 (w/v) of synthetic sea salts (Meersalz®, Heinsberg, Germany) in tap water. No food was provided during the 2 days of starvation. Substrates, the cofactor, and reagents were purchased from SigmaAldrich Chemical (St. Louis, MO, USA) and Merck (Darmstadt, Germany). Ceramic hydroxyapatite type I and an IPG strip (pH 5–8) were purchased from Bio-Rad laboratories (CA, USA). Sephacryl S-100, DEAE-Sepharose, Blue-Sepharose 6 fast flow, and Sephadex G-25 columns were purchased from GE Healthcare (Central Plaza, Singapore). 2.2. Methods 2.2.1. Isolation of enzymes Foot muscles from 2 kg of freshly killed hard clams were homogenized in a ratio of 1:5 (w/v) with cooled homogenization buffer containing 50 mM imidazole–HCl (pH 7) and 1 mM dithiothreitol (DTT) on ice. Homogenates were centrifuged at 12,000 × g for 20 min, and the supernatant was collected as the crude extract. The resulting supernatant was fractionated by ammonium sulfate precipitation to 55%–70% saturation. The precipitate was stirred for 60 min on ice and then centrifuged as described above. The final pellet was suspended in an adequate volume of homogenization buffer and passed through a Sephacryl S-100 column (2.6 × 100 cm) preequilibrated with homogenization buffer with 100 mM NaCl. SDH collected from this column was then applied to a ceramic hydroxyapatite column (1.6 × 20 cm) pre-equilibrated with CHT buffer containing 10 mM K2HPO4 (pH 7) and 1 mM DTT. The column was eluted with a linear gradient of 10–150 mM K2HPO4 in CHT buffer. The active SDH fractions were combined and concentrated in an Amicon cell (PM 10 membrane) and passed through a Sephadex G-25 column to remove the salts. The desalted sample was then applied to a DEAESepharose column (1.6 × 20 cm) pre-equilibrated with DEAE buffer containing 10 mM Tris–HCl (pH 7) and 1 mM DTT. The column was eluted with a linear gradient of 0–200 mM NaCl in DEAE buffer. The active SDH fractions were combined, concentrated in an Amicon cell,

39

and passed through a Sephadex G-25 column. The desalted sample was then applied to a Blue-Sepharose 6 fast flow column (1.6 × 20 cm) pre-equilibrated with Blue-Sepharose buffer containing 30 mM NaHCO3 (pH 6.5) and 1 mM DTT. The column was eluted with a linear gradient of 0–600 mM NaCl in Blue-Sepharose buffer. The active SDH fractions were combined and concentrated in an Amicon cell. The concentrated sample was supplemented with 30% glycerol and stored in a − 60 °C freezer. 2.2.2. Identification of enzymes Sodium dodecylsulfate (SDS) polyacrylamide electrophoresis (PAGE) was performed according to the method of Lane (1978) using 10% acrylamide gels. Sample and standard proteins were incubated at 100 °C in a ratio of 1:5 (v/v) with sample buffer containing 62.5 mM Tris–HCl, 2% SDS, 10% (v/v) glycerol, 0.1 M DTT, and 0.01% bromophenol blue (pH 6.8) for 5 min. The standard marker consisted of the following proteins (relative molecular mass in parenthesis): α-lactalbumin (14,400), trypsin inhibitor (20,100), carbonic anhydrase (30,000), ovalbumin (45,000), albumin (66,000), and phosphorylase b (97,000). The molecular weight of native SDH was determined by a Sephacryl S-200 column pre-equilibrated with Sephacryl S-200 buffer containing 0.25 M Tris–acetate (pH 7.4) and 100 mM NaCl. The standard marker consisted of the following proteins (relative molecular mass in parenthesis): aldolase (158,000), conalbumin (75,000), ovalbumin (43,000), and cytochrome c (12,384). Isoelectric point determinations were made by isoelectric focusing, using the IPG strip (pH 5–8) from Bio-Rad. Focusing was carried out according to the procedure recommended by the Bio-Rad literature using a Protean IEF Cell apparatus. Staining for both the SDS-PAGE and IEF gel was carried out according to the method of Merril et al. (1981). 2.2.3. Kinetics of the enzymes The activity of SDH was measured by monitoring the rate of enzymatic conversion of NADH to NAD+ at 340 nm using a Hitachi U3100 spectrophotometer equipped with an online data processing computer system (Tokyo, Japan) at 25 °C. One activity unit was defined as the amount of enzyme oxidizing 1 μmol of NADH per min at 25 °C. The standard assay conditions based on the method of de Zwaan and Zurburg (1981) were: 100 mM triethanolamine–HCl (pH 7.5), 1 mM KCN, 5 mM pyruvate, 0.24 mM NADH, and 100 mM glycine. Determination of lactate dehydrogenase (LDH) activity did not include amino acid substrates. The reaction was initiated by adding enzyme at 25 °C. The reaction was monitored for 1 min, and the reaction velocity was calculated using a time-scanning computer program. Michaelis constants, Km and Vmax, of SDH to various substrates [L-glycine (3.9, 7.8, 15.6, 31.3, 62.5, and 125 mM), L-alanine (3.9, 7.8, 15.6, 31.3, 62.5, and 125 mM), β-alanine (15.6, 31.3, 62.5, 125, and 250 mM), L-serine (7.8, 15.6, 31.3, 62.5, 125, and 250 mM), L-threonine (15.6, 31.3, 62.5, 125, 250, and 376 mM), L-2-aminobutyrate (2.5, 5, 10, 20, 30, and 50 mM), L-lysine (7.8, 15.6, 31.3, 62.5, 125, and 250 mM), L-arginine (7.8, 15.6, 31.3, 62.5, 125, and 250 mM), taurine (7.8, 15.6, 31.3, 62.5, 125, and 250 mM), pyruvate (0.3, 0.6, 1.25, 2.5, and 5 mM), 2-oxobutyrate (0.3, 0.6, 1.25, 2.5, 5, and 10 mM), 2-oxovalerate (3.1, 6.3, 12.5, 25, 50, and 100 mM), glyoxylate (2, 5, 10, 20, 40, and 60 mM), and α-ketoglutarate (3.1, 6.3, 12.5, 25, and 50 mM)] were determined from Lineweaver–Burk plots (Nelson and Cox, 2005). Kis of SDH were determined from Dixon plots with the effects of inhibitors (succinate, acetate, iminodiacetate, oxaloacetate, L-lactate, and D-lactate) with respect to glycine (Dixon, 1952). A hundred millimolar of MOPS buffer was used to replace 100 mM triethanolamine–HCl. 2.2.4. Effects of pH, temperature, ions, and storage time on the activity of SDH The effects of pH and temperature on the enzymatic activity were measured using 100 mM triethanolamine–HCl as a buffer system. The

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other conditions were the same as the standard assays except 20 min was used instead of 1 min of reaction time for the temperature experiment. The heat stability of SDH was determined by pre-incubating it at 56 °C for 20, 40, and 60 s. The other assay conditions were the same as those of the standard assays. The effects of ions on SDH activity were determined by 5 min of pre-incubation at 25 °C, and the reaction was initiated by adding NADH. The reaction velocity was calculated using a time-scanning computer program. The other assay conditions were the same as those of the standard assay. The enzymes were stored in a buffer containing 50 mM NaHCO3 (pH 7.5), 0.01% NaN3, and 1 mM DTT with three concentrations of (NH4)2SO4 (of 0.8, 1.6, and 2.18 M) or glycerol (10%, 20%, and 30%) at 4 °C for various periods of time. The assay conditions of the activity were the same as those in the standard assays. Samples were precipitated by 10% trichloroacetic acid to remove the DTT which would interfere with the protein assay. The resulting precipitate was assayed by a BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Bovine serum albumin (BSA) was used as the standard. 3. Results 3.1. Purification of strombine dehydrogenase A typical purification of strombine dehydrogenase (SDH) is presented in Table 1. SDH was separated from a large amount of contaminated proteins through Sephacryl S-100 and ceramic hydroxyapatite columns (Fig. 1A, B). They were eluted with a concentration of 50–70 mM NaCl in a DEAE-Sepharose column and by a concentration of 180–220 mM NaCl in a Blue-Sepharose column (Fig. 1C, D). The enzyme was purified by about 478-fold to a final specific activity of 827 U (mg protein)− 1 and was judged to be homogenous by SDS-PAGE (Fig. 2A). The molecular weight, 46 kDa, of purified SDH in the SDS-PAGE was calculated using molecular-mass standard markers (Fig. 3A). The determination of the molecular weight of native SDH was carried out by Sephacryl S-200 gel filtration. One species, equivalent to 46.3 kDa, was found (Fig. 3B). This indicates that SDH is a monomeric protein. Two bands of purified SDH were shown at the positions of pH 6.83 and 6.88 in the IEF gel (Fig. 2B). This enzyme preparation was used throughout the study.

specificity of purified SDH was assessed in terms of apparent Km and Vmax values. The low Km (34.2 and 39.5 mM, respectively) and high Vmax (471 and 339 U/mg, respectively) values of glycine and L-alanine indicate that both are the preferred substrates of the enzyme. In terms of Km and Vmax values, L-serine with Km of 163 mM and Vmax of 328 U/mg is the third preferred substrate for the enzyme. No enzymatic activity was observed with other L-amino acids including L-lysine, L-arginine, and taurine. This indicates that those amino acids are not substrates of the enzyme. Similarly, pyruvate with low Km (1.39 mM) and high Vmax (625 U/mg) values among five kinds of keto acid substrates indicates that it is the preferred keto acid substrate for the enzyme. The purified SDH also showed a high affinity for 2-oxobutyrate (2.64 mM for Km), but the Vmax with 2-oxobutyrate (100 U/mg) as a substrate was less than that with glyoxylate (239 U/mg) as a substrate. No enzymatic activity was observed with α-ketoglutarate. 3.3. Inhibitors The inhibitory effects of succinate, acetate, iminodiacetate, oxaloacetate, L-lactate, and D-lactate on the enzyme were tested. The inhibitory types of these six inhibitors on SDH are judged by double-reciprocal (1/v vs 1/s) plots. They are either competitive or mixed-competitive inhibitors. The apparent inhibition constants (Ki) of these inhibitors are determined from Dixon plots (Supplemental data II). They are shown in Table 3. Iminodiacetate with the lowest Ki of SDH at both pH 6.5 and 7.5 was the strongest inhibitor among these six inhibitors, whereas L- and D-lactate, end products of anaerobiosis, showed relatively higher Ki values than the others. The inhibitory effects of acetate, L-lactate, and D-lactate on the activity of SDH were pH independent, while those of succinate, iminodiacetate, and oxaloacetate on the enzyme were pH dependent. Inhibition of the activity of SDH at pH 6.5 by succinate was more potent, while those of iminodiacetate and oxaloacetate were higher at pH 7.5. 3.4. Effects of pH, temperature, and pre-incubation time at 56 °C on the activity of SDH pH and temperature profiles of purified SDH are shown in Fig. 4A and B. The enzyme had pH and temperature optima of 7.4–7.6 and 45– 46 °C, respectively. The heat stability of purified SDH is shown in Fig. 4C. The enzyme is heat labile. The activity of the enzyme pretreated at 56 °C for 20 s only retained 20% of its original activity.

3.2. Kinetic constants 3.5. Ion effects The purified SDH followed Michaelis–Menten saturation kinetics for six kinds of amino acids and four kinds of keto acid substrates. The kinetic constants for SDH are calculated from double-reciprocal (1/v vs 1/s) plots (Supplemental data I). They are listed in Table 2. The substrate

Table 1 Summary of the purification of strombine dehydrogenase from the foot of the hard clam Meretrix lusoria. Step

Total activity (U)

Total protein (mg)

Specific activity (U/mg)

Yield (%)

Crude homogenate Ammonium sulfate fractionation Sephacryl S-100 chromatography Hydroxyapatite adsorption DEAE-Sepharose chromatography Blue-Sepharose chromatography

2160 ± 170 1360 ± 3.9

1250 ± 7.1 206 ± 2.7

1.73 ± 0.15 6.62 ± 0.10

100 63

Purification factor 1 3.8

1110 ± 26

12 ± 0.52

92.9 ± 1.9

51

14

741 ± 2.9

2.68 ± 0.42

279 ± 12

34

3

426 ± 82

0.6 ± 0.06

706 ± 61

20

2.5

246 ± 40

0.3 ± 0.05

827 ± 3.7

11

1.2

Data from two preparations are presented as the mean ± standard deviation.

The effects of ions on the activity of SDH are shown in Fig. 5. Basically, Na+ and K+ had no effect on the enzymatic activity. Calcium ions and Mg2+ had little effect on the activity of the enzyme. Approximately 10% and 20% of the enzymatic activity were inhibited by 30 and 50 mM Ca2+ and Mg2+, respectively (Fig. 5A). The effects of Cu2+ and Mn2+ on the enzymatic activity were higher than those of Ca2+ and Mg2+ (Fig. 5B). Fifty-five percent and 13% of the enzymatic activity were inhibited by 1 mM of Cu2+ and Mn2+, respectively. Ferric ions and Zn2+ had strong effects on the enzymatic activity. Half of the enzymatic activity was inhibited by 0.2 and 0.6 mM of Fe3+ and Zn2+. 3.6. Storage conditions at 4 °C The activity profiles of SDH stored at three concentrations of ammonium sulfate and glycerol at 4 °C are shown in Fig. 6. The enzymatic activity remained at 100% for 2 weeks when the enzyme was stored with 0.8 M of ammonium sulfate and 10%, 20%, and 30% glycerol at 4 °C. The activity of the enzyme in the control group was stable for 1 week, sharply declined to 60% after 2 weeks of storage, and was maintained at 40%–50% of its activity for the subsequent

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1.0

A Absorbance Activity

0.06

0.6

0.04

0.4

0.02

0.2

0.00 150 0.5

200

250

300

0.0 400 1.0

350

B

0.4

Absorbance280 nm

0.8

0.3

0.6

0.2

0.4

0.1

0.2

0.0 0.100 0

50

100

150

0.0 250 0.6

200

C NaCl

0.075

120

0.8

K2HPO4

80 40 0 200 150

0.4

100

0.050 0.2

50

0.025 0.000 0.10

50

100

150

0.0 250 10

200

D

0.08 0.06

6

0.04

4

0.02

2

0.00 20

40

0 600

8

NaCl

0

Concentration (mM)

0.08

Activity (U/ml)

0.10

41

60

80

100

120

0 140

400

200

0

Elution volume (ml) Fig. 1. Chromatography of strombine dehydrogenase on a Sephacryl S-100 column (A), ceramic hydroxyapatite column (B), DEAE-Sepharose column (C), and Blue-Sepharose column (D). Details are given in the Material and methods.

6 weeks of storage. A low concentration of ammonium sulfate (0.8 M) stabilized the activity of the enzyme for short-term storage (b3 weeks), while it had a negative effect on enzymatic activity with long-term storage (3–8 weeks). Generally, a high concentration of ammonium sulfate (2.18 M) had a harmful effect on enzymatic activity during storage. The addition of glycerol stabilized the enzyme for 4 weeks of storage. After that, the glycerol had no significant effect on the enzymatic activity. 4. Discussion In mollusks, both opine dehydrogenases and LDH are well known as terminal enzymes for regulating the cytosolic redox balance in glycolysis under anoxia (Dando et al., 1981). Generally, the opine and lactate pathways respectively predominate in less-mobile and phylogenetically lower animals with poorly developed organs and in much more-mobile and phylogenetically higher animals with welldeveloped organs (Sato et al., 1993). These characteristics were observed in the foot of the hard clam where the activity of LDH was only 5% that of opine dehydrogenase (Lee and Lee, in press). The activities of alanopine dehydrogenase (ADH), strombine dehydrogenase (SDH), and octopine dehydrogenase (ODH) were the highest

among opine dehydrogenases in the foot of the hard clam under normoxia and after anoxic exposure. SDH showed equivalent affinities for both glycine and alanine in the foot of the hard clam in this study and should play an important physiological role in its metabolism. SDH (Mr. 46,000) in the foot of the hard clam is a monomeric protein; in the literature, SDH was found in Aphrodite aculeate (Mr. 44,000) (Storey, 1983), SDH in Mytilus edulis (Mr. 40,000) (De Zwaan and Zurburg, 1981), ADH in Littorina littorea (Mr. 42,200) (Plaxton and Storey, 1982), ADH in C. gigas (Mr. 47,000) (Fields and Hochachka, 1981), ODH in C. lacteus (Mr. 40,000) (Gäde and Carlsson, 1984), TDH in A. iricolor (Mr. 43,500) (Kanno et al., 1996), and BADH in C. grata (Mr. 40,000) (Kanno et al., 1999). All these results clarify that opine dehydrogenases are a family of monomeric proteins with Mr. values of 40,000–47,000. However, this rule has its exceptions in the demosponge S. domuncula SDH which is a dimer (Plese et al., 2009). pIs of SDH in the foot of the hard clam were higher than that (4.75–4.8) of SDH in M. mercenaria (Fields and Storey, 1987), and that (5.4–5.6) of ODH in C. lacteus. In A. iricolor like for SDH in the hard clam, two species of TDH, equivalent to pI 6.4 and 7.5, were found in the IEF gel (Kanno et al., 1996). SDH has a high specificity for pyruvate compared to the other keto acids tested. Comparisons of the apparent Vmax values obtained indicate

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Fig. 2. SDS-gel electrophoretogram (A) of samples after (2) crude extraction (0.142 μg), (3) ammonium sulfate fractionation (0.133 μg), (4) Sephacryl S-100 column (0.288 μg), (5) hydroxyapatite column (0.246 μg), (6) DEAE-Sepharose column (0.213 μg), and (7) Blue-Sepharose 6 fast flow column (0.21 μg). The protein standards (α-lactalbumin, trypsin inhibitor, carbonic anhydrase, ovalbumin, albumin, and phosphorylase b) are shown in (1, 0.288 μg) and (8, 0.288 μg). Isoelectric focusing electrophoretogram using an IPG strip (pH 5–8) (B) of purified strombine dehydrogenase. The positions of the enzyme are indicated by arrows. The gel was stained for proteins by the silver staining method.

that the methyl group in the substrate is important for substrate catalysis. Either the removal of this group (glyoxylate) or the addition of this group (2-oxobutyrate) and an ethyl group (2-oxovalerate) leads to a decrease in the apparent Vmax. However, no change in the apparent Km by the addition of a methyl group (2-oxobutyrate) indicates that the inclusion of this group to pyruvate did not affect the affinity of the enzyme for the substrate. Both the removal of this group (glyoxylate) and the addition of an ethyl group (2-oxovalerate) lead to a great increase in the apparent Km. However, the removal of the methyl group from pyruvate did not change the affinity of glyoxylate to SDH (Km = 4.18 mM) of M. mercenaria or that (3.49 mM) of adductor muscle in C. gigas as much to that (58.6 mM) of the hard clam (Fields and Hochachka, 1981; Fields and Storey, 1987). The affinity and catalysis of purified SDH of the hard clam for glycine being comparable to those for alanine indicated that this Table 2 Apparent Km and Vmax values for Meretrix lusoria strombine dehydrogenase with different amino and keto acids. Substrate

Fig. 3. Plots of Rf against the log of Mr corresponding to the determination of molecular weight of purified strombine dehydrogenase (*) by SDS-PAGE (A). The corresponding Rf values were calculated by dividing the migration distance of the respective proteins by that of the dye marker. The protein standards were (1) α-lactalbumin, (2) trypsin inhibitor, (3) carbonic anhydrase, (4) ovalbumin, and (5) albumin. Plots of Kav against the log of Mr corresponding to the determination of the mol. wt of purified strombine dehydrogenase (*) by a Sephacryl S-200 column equilibrated with 0.25 M Tris–acetate (pH 7.4) and 100 mM NaCl (B). The protein standards were (1) cytochrome c, (2) ovalbumin, (3) conalbumin, and (4) aldolase. The voided volume was determined by the elution volume of blue dextran 2000. Kav = (Ve − Vo)/(Vt − Vo) where Ve is the elution volume for the protein, Vo is the column void volume, and Vt is the total bed volume.

Amino acids Glycine L-Alanine β-alanine L-Serine L-Threonine L-2-Aminobutyrate L-Lysine L-Arginine Taurine Keto acids Pyruvate 2-Oxobutyrate 2-Oxovalerate Glyoxylate α-Ketoglutarate

Apparent Km (mM)

Apparent Vmax (μmol min− 1 mg protein− 1)

34.2 39.5 196 163 303 45.9 – – –

471 339 76.3 328 74.6 271 – – –

1.39 2.64 68 58.6 –

625 100 84 239 –

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100

Table 3 Apparent inhibition constants from Dixon plots of Meretrix lusoria strombine dehydrogenases for succinate, acetate, iminodiacetate, oxaloacetate, L-lactate, and D-lactate at pH 6.5 and 7.5. Ki (mM)

Succinate Acetate Iminodiacetate Oxaloacetate L-Lactate D-Lactate

pH 6.5

pH 7.5

3.98 5.57 1.35 14.3 21.2 16.4

15.4 3.69 0.63 6.93 22.9 17

1.6

A

Ca2+ Mg2+ Na+ K+

60

enzyme exhibits activity not only for SDH but also for ADH. This observation was supported by Fields (1976) who proposed that the same polypeptide might contribute to the activities of SDH and ADH in the oyster adductor muscle. The specificity of SDH for amino acids varies with the position of the amino group and the presence of a hydroxyl group of amino acids. The position of the amino group in α-carbon is very important in substrate binding and catalysis. A higher Km and a lower Vmax were found for β-alanine due to an amino group in the β-carbon. The presence of a hydroxyl group in L-serine and L-threonine significantly increased the apparent Km,

40 20 0 100

0

10

20

30

40

50

B 80 60

Fe3+ Zn2+ Cu2+ Mn2+

40 20 0 0.0

0.2

0.4

0.6

20.0

40.0

Concentration (mM) Fig. 5. Effects of Ca2+, Mg2+, Na+, and K+ (A) and Fe3+, Zn2+, Cu2+, and Mn2+ (B) on the activity of purified strombine dehydrogenase.

A

but only a hydroxyl group in L-threonine decreased the apparent Vmax. However, the addition of an ethyl group to glycine (L-2-aminobutyrate) did not change the apparent Km. Kinetic properties of SDH of the hard clam to L-serine and L-2-aminobutyrate were also observed in ADH of the oyster C. gigas (Fields and Hochachka, 1981). The size of the R-group of the amino acids is also an important parameter for the activity of SDH. SDH of the hard clam cannot utilize L-lysine and L-arginine with a larger R-group as substrates.

1.4

1.2

Activity (units/ml)

80

Percentage of original activity (%)

Inhibitors

43

1.0

0.8 6.5

7.0

7.5

8.0

8.5

9.0

pH 0.6

Control

100

B

0.8 M (NH4)2SO4

A

80

1.6 M (NH4)2SO4 2.18 M (NH4)2SO4

60

0.4

40

0.0

Percentage of original activity (%)

20

30

40

50

60

Temperature ( ) 100

C

80

Residual activity (%)

0.2 20 0 100

B 80 60

60 40

Control 10% glycerol 20% glycerol 30% glycerol

40 20

20 0

10

20

30

40

50

60

Incubation time (seconds) Fig. 4. Effects of pH (A), temperature (B), and pre-incubation time at 56 °C (C) on the activity of purified strombine dehydrogenase.

0 0

1

2

3

4

5

6

7

8

Storage time (weeks) Fig. 6. Effects of storage time on the activity of purified strombine dehydrogenase stored with ammonium sulfate (A) and glycerol (B).

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A.-C. Lee et al. / Comparative Biochemistry and Physiology, Part B 158 (2011) 38–45

Among the metabolites of succinate, acetate, iminodiacetate, oxaloactetate, L-lactate, and D-lactate; iminodiacetate, an analogue of strombine, showed the most potent inhibition on the activity of SDH in the hard clam. The great inhibition of iminodiacetate on the activity of opine dehydrogenases was also found in ADH and SDH of the sea mouse A. aculeata (Polychaeta) (Storey, 1983). Succinate, an end product of anaerobiosis, also showed an inhibitory effect on the activity of SDH, and this inhibition was pH dependent. The Ki (3.98 mM) of succinate at pH 6.5 being lower than that (15.4 mM) at pH 7.5 indicates that the inhibition of the activity of SDH by succinate is more potent in acidic environments. It was also reported that succinate with no effect on the activity of ADH of the common periwinkle L. littorea at pH 7.5 was inhibitory at high concentrations at pH 6.5 (Plaxton and Storey, 1982). A concentration of 8 μmol/g wet weight (being equal to 10 mM) accumulated in the foot of the hard clam after 3 days anoxic exposure at 30 °C (Lee et al., 2008). Therefore, the activity of SDH in the foot of the hard clam should be inhibited by succinate under anaerobic metabolism. It was also found that succinate had no effect on the activities of ADH in the foot of L. littorea at pH 7.5, but it had an inhibitory activity at pH 6.5 (Plaxton and Storey, 1982). Similarly, the apparent Ki of ADH in oyster adductor muscle for succinate was found to decrease from 22 mM at pH 7.5 to 1.8 mM at pH 6.5 (Fields and Hochachka, 1981). The physiological roles of SDH vary in different species of organisms. The imino acids, alanopine and strombine, were not produced in the oyster C. virginica during 96 h of anoxia, but they accumulated during 12 h of the recovery period (Eberlee et al., 1983). However, these opines accumulated in the adductor muscle of the sea mussel M. edulis after 48 h of shell valve closure and their concentration persistently increased even after the recovery period (de Zwaan and Zurburg, 1981; de Zwaan et al., 1983). Whether the hard clam exposed to anoxia produces acetic acid is unclear, but it is an end product of anaerobiosis in the sea mussel M. edulis (Kluytmans et al., 1978; van den Thillart and de Vries, 1985). The inhibitory activity of acetate on SDH was pH independent and comparable to that of succinate since the apparent Ki values for both are very close. The activity of L-LDH of the hard clam was b5% of opine dehydrogenase activity (Lee and Lee, in press). The role of its product, L-lactate, on regulating anaerobic metabolism of the hard clam should be minor. A higher value of Ki (22 mM) of L-/D-lactate for SDH was obtained in this study. Weak inhibition of L-/D-lactate (b10% inhibition at 20 mM) on the activity of pharynx SDH of the sea mouse A. aculeata was also reported (Storey, 1983). However, L- and D-lactate, which had much greater inhibitory effect on the activity of muscle ADH in A. aculeata with respect to L-alanine, were weaker inhibitors with respect to pyruvate (Storey, 1983). However, L-lactate with a small value of Ki (0.9–3.15 mM) potently inhibits the activity of ADH of L. littorea at both pH 6.5 and 7.5 (Plaxton and Storey, 1982). The ionic components of seawater were found to affect the digging ability of hard clams (Lee et al., 2007). Their anaerobic metabolism was induced by the removal of magnesium ions from seawater. The activity of ADH from the foot muscle of L. littorea was inhibited by monovalent and divalent ions. Fifty millimolar of MgCl2 inhibited 45% of the activity of ADH (Plaxton and Storey, 1982). However, effects of salt on the activities of muscle ADH and pharynx SDH of A. aculeata were not found (Storey, 1983). In this study, 20% of SDH activity was inhibited by 50 mM of either CaCl2 or MgCl2. Surprisingly, the inhibitory effect of Fe3+ on the activity of SDH was the highest among these metal ions tested. The concentration of FeCl3 in Walne's medium was 5 μM. Thirteen percent of SDH activity was inhibited by 20 μM FeCl3 in this study. Therefore, the functional anaerobic metabolism of the hard clam might be disturbed by the residual Fe3+ from microalgae cultured with Walne's medium or other media containing Fe3+. Supplementary materials related to this article can be found online at doi: 10.1016/j.cbpb.2010.09.003.

Acknowledgements The authors are grateful for financial support from the National Science Council of Taiwan (NSC97-2313-B-415-003-MY3). The authors also thank Dr. H.F. Liao in the Department of Biochemical Science and Technology for assisting us in the determination of isoelectric point of SDH. References Barrett, J., Butterworth, P.E., 1981. A novel amino acid linked dehydrogenase in the sponge Halichondria panacea (Pallas). Comp. Biochem. Physiol. B 70, 141–146. Dando, P.R., Storey, K.B., Hochachka, P.W., Storey, J.M., 1981. Multiple dehydrogenases in marine molluscs: electrophoretic analysis of alanopine dehydrogenase, strombine dehydrogenase, octopine dehydrogenase and lactate dehydrogenase. Mar. Biol. Lett. 2, 249–257. de Zwaan, A., 1983. Carbohydrate catabolism in bivalves. In: Hochachka, P.W. (Ed.), Metabolic Biochemistry and Molecular Biomechanics. : The Mollusca, I. Academic Press, New York, pp. 137–175. 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