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Metabolic responses and arginine kinase expression under hypoxic stress of the kuruma prawn Marsupenaeus japonicus
Comparative Biochemistry and Physiology, Part A 146 (2007) 40 – 46 www.elsevier.com/locate/cbpa
Metabolic responses and arginine kinase expression under hypoxic stress of the kuruma prawn Marsupenaeus japonicus Hiroki Abe ⁎, Shun Hirai, Shigeru Okada Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan Received 4 April 2006; received in revised form 10 August 2006; accepted 18 August 2006 Available online 30 August 2006
Abstract In response to hypoxia at PO2 1.3–1.7 mg/L for 6 h, the kuruma prawn Marsupenaeus (Penaeus) japonicus showed a dramatic decrease in phosphoarginine storage in muscle, with normal levels restored during 4-h post-hypoxic recovery. Large stores of muscle glycogen only decreased between 4 and 6 h during hypoxia, but greatly diminished during recovery. Muscle ATP levels and energy charge decreased only slightly under hypoxia. Lactate levels increased slightly during hypoxia and promptly returned to control levels during recovery. These data indicate that phosphoarginine works in muscle as an ATP buffer during hypoxia and glycogen is utilized as an energy source during recovery. Under hypoxia, up- and down-regulated proteins were identified after 2D electrophoresis and partial sequences were obtained after protease digestion. Fructose bisphosphate aldolase was down-regulated during hypoxia, suggesting the suppression of glycolysis under hypoxia. Several partial sequences from three protein spots up-regulated under hypoxia were all assigned to arginine kinase, suggesting the existence of several isoforms of arginine kinase in the muscle of M. japonicus. This arginine kinase up-regulation under hypoxia may indicate a provision for oxygen re-supply after anaerobiosis. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia. © 2006 Elsevier Inc. All rights reserved. Keywords: Arginine kinase; Phosphoarginine; ATP; Energy metabolism; Glycogen; Hypoxia; Kuruma prawn; Marsupenaeus japonicus
1. Introduction Aquatic invertebrates encounter periodic environmental hypoxia or anoxia and evolve effective anaerobic mechanisms to cope with reduced ambient oxygen concentrations (Hochachka, 1980; Hochachka and Somero, 1984). In contrast to well-known hypoxia-tolerant invertebrates such as bivalve mollusks and annelids, hypoxia tolerance appears to be highly species-specific in crustaceans (Schmitt and Uglow, 1998). In decapod crustaceans, burrowing species usually show higher hypoxia tolerance than free-swimming species. The burrowing and hypoxia-tolerant decapods often maintain large stores of energy sources such as glycogen and phosphoarginine and reduce their metabolic rate under hypoxia (Hervant et al., 1999). During anaerobiosis, crustaceans have been considered to employ only classical anaerobic glycolysis, leading to L-lactate as the sole end⁎ Corresponding author. Tel.: +81 3 5841 5296; fax: +81 3 5841 8166. E-mail address: [email protected] (H. Abe). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.08.027
product, whereas in some species alanine and succinate have also been reported to be accumulated (Zebe, 1982; Onnen and Zebe, 1983; Gäde, 1984; Hill et al., 1991). We also confirmed that the crayfish Procambarus clarkii accumulated D- and L-alanine besides L-lactate in muscle and hepatopancreas under severe hypoxia below 0.1 mg O2/L (Fujimori and Abe, 2002). Under severe environmental hypoxia, crustaceans need to regulate the production of some key enzyme proteins to survive in harsh environments, as is known in other animals (Hochachka and Somero, 2002). There have been only a few reports so far on gene and protein expression in crustaceans during hypoxia: heat shock/α-crystallin protein, p26, in Artemia franciscana embryo (Willsie and Clegg, 2001); heat induced factor mediated globin gene in Daphnia magna (Gorr et al., 2004); and several genes including Mn-superoxide dismutase gene and hemocyanin protein in blue crab Callinectes sapidus (Brouwer et al., 2004). It is of particular interest to clarify the regulation of gene and protein expression levels in relation to energy metabolism under oxygen-limiting conditions.
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The kuruma prawn Marsupenaeus (Penaeus) japonicus is a swimming and burrowing species and the most commercially valuable penaeid species that is widely cultured in Japan and other Asian countries. The prawn is transported live to markets and consumers in dry sawdust at low temperature (just above 10 °C) and is kept alive for several days. There are a few reports in the literature on the hypoxia tolerance of this species during aerial exposure (Furusho et al., 1988; Paterson, 1993a,b; Chen and Chen, 1998), but no report on the metabolic responses of M. japonicus in relation to protein over-expression during hypoxia. The objective of this study was to obtain information on the metabolic responses of M. japonicus under hypoxic stress and during post-hypoxic recovery, and on up- and down-regulated proteins during hypoxia. 2. Materials and methods 2.1. Animals and hypoxia stress loading Live adult M. japonicus weighing approximately 20 g were obtained from a culture farm in Miyazaki Prefecture, Japan and transported live by air to the laboratory in dry sawdust at low temperature. They were reared in a 60-L aquarium supplied with aerated circulating seawater (filtrated natural seawater of 35 ppt) and maintained at 15 °C. At the bottom of the aquarium was a fine sand layer of 10 cm in depth. The animals were fed daily with dry pellets for prawns, but were not fed for 2 days prior to the experiments. After acclimation to the aquarium conditions, the animals were exposed to hypoxia (PO2 = 1.3–1.7 mg/L, ∼3 kPa) for 6 h under constant bubbling of nitrogen gas. The water surface was covered with a plastic sheet during hypoxia and the sand layer was removed. After hypoxia, the animals were recovered for 4 h in hyperoxic conditions with vigorous aeration. During hypoxia and recovery, 4 to 5 individuals were taken out at 2-h intervals. Special care was taken to avoid tail flips by prawn during sampling. After decapitation, the tail muscle and hepatopancreas were excised and quickly frozen in liquid nitrogen. A perchloric acid extract was prepared from a 1-g sample as previously described (Okuma and Abe, 1994b). All prawns used in the experiment were in the intermolt stage. 2.2. Analytical methods Free amino acid analysis was performed on an amino acid autoanalyzer (L-8500A; Hitachi, Tokyo, Japan). Phosphoarginine was determined by high-performance liquid chromatography (HPLC). The HPLC system (Jasco, Tokyo, Japan) consisted of a PU-1580 pump, a DG-1580-53 line degasser, an LG-158002 ternary gradient unit, a CO-1565 column oven, an AS-2057 autosampler with a cooling system, a UV-1570 UV–VIS detector, and an 807-IT integrator. An anion-exchange column (Shim-pack CLC-NH2; Shimadzu, Kyoto, Japan) was used with a guard column. Elution was conducted isocratically with 10 mM sodium phosphate buffer, pH 2.6, containing 20% acetonitrile at a flow rate of 1 mL/min at 40 °C. Phosphoarginine was detected at 205 nm.
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Nucleotides were determined on the above HPLC system essentially according to the method of Okuma and Abe (1994a). Muscle glycogen and L-lactate were determined enzymatically according to the methods of Keppler and Decker (1984) and Noll (1984), respectively. All chemicals were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO, USA) or Wako Pure Chemical Industries (Osaka, Japan) unless otherwise stated. 2.3. Protein extraction for two-dimensional (2D) electrophoresis Muscle tissue was homogenized with a Polytron homogenizer (Kinematica, Littau-Lucerne, Switzerland) in 4 volumes of 50 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 10 000 ×g for 30 min. Proteins in the supernatant were salted out with ammonium sulfate between 30% and 70% saturation. The precipitate after centrifugation as above was dissolved in 5 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 0.2 mM PMSF and dialyzed overnight against the same buffer. After centrifugation as above, the supernatant obtained was subjected to 2D electrophoresis. Protein concentration was determined with a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). 2.4. Analytical 2D electrophoresis and peptide mapping with limited proteolysis Analytical 2D electrophoresis was performed using the method of O'Farrell (1975). The first dimensional isoelectric focusing was conducted using 4% polyacrylamide gel containing 8 M urea, 2% Triton X-100, and 1.6% and 0.4% Ampholine (Pharmacia, Uppsala, Sweden), pH 5–8 and 3–10, respectively. The second dimensional sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) was performed on an 11.5% slab gel. After electrophoreses, the gel was stained with 0.25% Coomassie brilliant blue R-250. Electrophoretic patterns were analyzed with an ImageMaster VDS-CL (Amersham Bioscience, Cleveland, OH, USA). After 2D electrophoresis, proteins up- or down-regulated after 6-h hypoxia were cut out from the gel and equilibrated in 0.125 M Tris–HCl, pH 6.8, containing 0.1% SDS for 30 min with shaking. The electrophoresis was repeated over 10 times and the protein spots of interest were pooled and concentrated. Each protein was subjected to limited proteolysis with Staphylococcus aureus V8 protease (Wako) during SDS-PAGE according to the method of Cleveland et al. (1977). 2.5. Determination of amino acid sequence of peptide fragments The proteins on Coomassie-stained SDS-PAGE gels were electrically transferred onto Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and subjected to a protein sequencer (Applied Biosystem model 492HT; Foster City, CA, USA). The amino acid sequences of peptide fragments were compared using a BLAST search.
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Fig. 1. Changes in phosphoarginine (A) and glycogen (B) levels in the muscle and hepatopancreas of the kuruma prawn M. japonicus in 35 ppt seawater at 15 °C during hypoxia at PO2 1.3–1.7 mg/L (around 3 kPa) for 6 h and subsequent recovery for 4 h. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 compared with 0 time control (n = 4–5).
2.6. Statistical analyses The results of metabolite analyses are presented as mean ± S.D. (n = 4–5). The data were analyzed statistically using one-way ANOVA followed by post-hoc comparison of means (LSD test). In all comparison, 0 time value was used as control. 3. Results 3.1. Biochemical responses to hypoxia and recovery Under hypoxia at PO2 values between 1.3 and 1.7 mg/L (approx. 3 kPa) for 6 h, only three animals died between 4 and 6 h and the survival rate was 90%. The large store of
phosphoarginine accumulated in the muscle of control prawn (31 μmol/g wet wt., n = 5) decreased dramatically to one-fifth (6.1 μmol/g wet wt., n = 4–5) during hypoxia and increased linearly to the control level after 4 h of recovery (Fig. 1A). This was also the case in hepatopancreas, where the concentration of phosphoarginine was far lower than that in muscle. Muscle glycogen levels decreased during the last 2 h of hypoxia and the decrease was significant statistically (Fig. 1B). A much greater reduction in muscle glycogen level was found during recovery. In hepatopancreas, the glycogen level was only half of that in muscle and decreased significantly during the last 2 h of hypoxia. In contrast to muscle, glycogen levels in hepatopancreas increased to the control level during recovery.
Fig. 2. Changes in nucleotide levels in the muscle (A) and hepatopancreas (B) of M. japonicus during hypoxia and recovery. Refer to the legend of Fig. 1 for experimental conditions and significances.
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increased significantly and stoichiometrically with the ATP decrease during hypoxia (Fig. 2B). Under hypoxia, L-lactate increased in muscle and returned to the control level after 2 h of recovery (Fig. 3). However, the increase in lactate during hypoxia (2.3 μmol/g wet wt.) was far lower than the decrease in glycogen (10 μmol glucose unit/g wet wt.) in muscle (Fig. 1B). Of all amino acid determined, only D- and L-alanine increased significantly from control level (12.5 μmol/g) to 18.9 μmol/g during 6-h hypoxia (data not shown). 3.2. Changes in protein expression under hypoxic stress
Fig. 3. Changes in L-lactate in the muscle of M. japonicus during hypoxia and recovery. Refer to the legend of Fig. 1 for experimental conditions and significances.
Fig. 2 shows the changes in nucleotides during hypoxia and recovery. Muscle ATP levels decreased moderately during hypoxia and then increased back to the control level during recovery. As a result, the adenylate energy charge was maintained between 0.84 and 0.97 in muscle during hypoxia and recovery. The lower ATP levels in hepatopancreas also decreased during hypoxia and returned to the control level during recovery. In hepatopancreas, however, the energy charge was as low as 0.7 even in control animals, and declined to 0.5 during hypoxia. Along with the decrease in ATP in hepatopancreas, AMP levels
Fig. 4 shows the 2D electrophoretic profiles of muscle protein solutions from control and 6-h hypoxic individuals of M. japonicus (250 μg each). Many of the protein spots were upor down-regulated under hypoxia, but four spots (nos. 1–4) were selected in the present experiment based on the reproducibility and differences of the spots between control and hypoxic individuals (Fig. 4C–F). From protein spot 1, for which expression decreased to onefifth under hypoxia compared with control, three partial amino acid sequences (1a–c) were obtained (Table 1). Of these sequences, 1b was assigned to fructose bisphosphate aldolase (EC 4.1.2.13) of crucian carp Carassius auratus, mouse Mus musculus, and human Homo sapiens using a BLAST search. Sequence 1b showed 87% amino acid identity to these aldolases. Sequences, 1a and 1c, showed no matches with the BLAST database. All other partial amino acid sequences from proteins, 2, 3, and 4, of which expressions increased 2- to 5-fold under hypoxia, were assigned to arginine kinases (EC 2.7.3.3)
Fig. 4. Two-dimensional electrophoretic profiles of proteins from the muscle of control M. japonicus (A) and that exposed to 6-h hypoxia (B). Arrows with numbers indicate the protein spots down- or up-regulated under hypoxia compared with the control. Panels C and D and those E and F are magnified photos in the related regions of panels A and B, respectively.
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Table 1 Partial amino acid sequences of peptides obtained from the protein spots 1–4 in Fig. 4 which were down- or up-regulated under hypoxic stress of M. japonicus Peptide no.
Partial amino acid sequence
1a 1b
NAKKYRQLKFTADKAFR LSQRYAQYKKDGADFAIKKYDDL
1c 2a
STTQGLDDLGQQQGAAILPNFI AYTLFAPLFDPIIED
2b 3a 3b
DYAVGFKQTDKKPNK AELKGTYFPKTYMSKGQTD VQQKLIDDHFLFKEG
4a 4b
VQQKLIDDHFLFKEG AQYKEMETKVSSTLSMEVSSEL
4c 4d
KELKGLYFKVSGMKAQYETALG AYTLFAPLFDPIIED
Homologous proteins (sources)
Identity (%)
Fructose bisphosphate aldolase (Carassius auratus, AAA84887) Fructose bisphosphate aldolase (Mus musculus, AAA37210) Fructose bisphosphate aldolase (Homo sapiens, CAA30979)
87 87 87
Arginine Arginine Arginine Arginine
100 100 100 86
kinase (Marsupenaeus japonicus, P51545) kinase (Pachygrapsus marmoratus, AAG01175) kinase (Callinectes sapidus, AAF43436) kinase (Marsupenaeus japonicus)
Arginine kinase (Marsupenaeus japonicus) Arginine kinase (Pachygrapsus marmoratus) Arginine kinase (Eriocheir sinensis, AAF43437) Same as 3b Arginine kinase (Drosophila melanogaster, P48610) Arginine kinase (Eriocheir sinensis) Arginine kinase (Carcinus maenas, AAD48470)
100 100 100 93 93 93
Same as 2a
Homologous proteins were obtained from the BLAST database. GenBank accession numbers are shown after each species name.
from invertebrates, including M. japonicus, with high amino acid identities of 86–100% (Table 1). All of these invertebrates are crustaceans, except for fruit fly Drosophila melanogaster, and all are crab species, except for M. japonicus. Several partial sequences obtained from different proteins showed the same
sequences, as observed for 2a and 4d, and 3b and 4a. On the other hand, 3a and 4c matched with no protein sequences. Fig. 5 shows the amino acid sequences of arginine kinase from M. japonicus and three crab species, as well as partial sequences obtained in the present experiment. The amino acid
Fig. 5. Alignment of deduced amino acid sequences of arginine kinases from representative crustaceans with partial sequences (in bold letters) identified in the muscle of M. japonicus under hypoxia. Asterisks indicate the identical amino acid residues among the four sequences. In parentheses, sequence numbers shown in Table 1 are indicated. Residues interacting with the amino and carboxyl groups of substrate arginine are boxed. Aspartate and arginine residues, key residues in arginine kinase function, are boxed by dotted line (Suzuki et al., 2000). Refer to Table 1 for accession number for each sequence.
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sequences of crustacean arginine kinase show high homology and four amino acid residues interacting with the substrate arginine are conserved in all crustacean enzymes. Two additional residues, aspartate and arginine, are also conserved and are supposed to be essential for enzyme function (Strong and Ellington, 1995; Suzuki et al., 2000). In the partial sequences obtained in the present experiment, sequences 2a (4d) and 3b (4a) coincided completely with those of crustacean arginine kinases. Other partial sequences, 2b and 4b, were also similar to the crustacean sequences, but showed several amino acid substitutions. 4. Discussion When M. japonicus was subjected to severe hypoxia below 0.1 mg O2/L under bubbling of nitrogen gas, the survival rate was extremely low even during 3-h hypoxia (data not shown). Thus, M. japonicus is more oxygen-sensitive than crayfish P. clarkii which easily survives for at least 12 h under the same conditions (Fujimori and Abe, 2002). Based on these results, PO2 was set at approximately 3 kPa in the present study. This PO2 level may occur in shallow coastal regions during summer and was known to be avoided by some Penaeus prawns (Renaud, 1986). The muscle phosphoarginine concentration was extremely high, 31 μmol/g, in normoxic M. japonicus and dramatically decreased to 6.1 μmol/g during hypoxia (Fig. 1A). This phosphoarginine level was 1.5-fold higher than that in crayfish Orconectes limosus (Gäde, 1984). In contrast, muscle ATP levels only moderately decreased under hypoxia (Fig. 2A). These data suggest that the ATP level in muscle was effectively buffered by phosphoarginine. The muscle glycogen level decreased only between 4 and 6 h during hypoxia (Fig. 1B). Thus, in M. japonicus under hypoxia of ∼3 kPa for 6 h, muscle energy may be supplied mainly by phosphoarginine, followed by glycogenolysis. The level of muscle glycogen was almost the same as that in crayfish O. limosus (Gäde, 1984) and P. clarkii (Fujimori and Abe, 2002). Muscle glycogen decreased only during the last 2 h of hypoxia and largely during recovery (Fig. 1B). This suggests that unlike P. clarkii, M. japonicus mainly depends on glycogenolysis during recovery from hypoxia, which leads to prompt recovery of phosphoarginine and ATP levels (Figs. 1A and 2A). No increase in glucose was found throughout hypoxia and recovery periods (data not shown). This is consistent with the case of spiny lobster Panulirus interruptus under hypoxia (Ocampo et al., 2003). The muscle ATP level was almost the same as that reported previously in this species (Furusho et al., 1988; Paterson, 1993b) but the decrease of ATP during hypoxia was far lower than that during aerial exposure in dry sawdust in these reports, although the experimental period was longer in aerial exposure (24 h) than hypoxia in the present study (6 h). These data may suggest that some different anaerobic mechanisms work between aerial exposure and environmental hypoxia. In hepatopancreas, phosphoarginine and glycogen levels were far lower than in muscle and decreased during hypoxia, suggesting that ATP buffering by phosphoarginine is not effective in hepatopancreas. The content of ATP and energy
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charge were also very low in hepatopancreas and both lowered during hypoxia. Thus, the energy state and utilization of hepatopancreas in normoxic and hypoxic water may be quite different from those of muscle. Under hypoxia, the increase in muscle lactate was limited to 2.3 μmol/g wet wt., whereas the decrease in glycogen was 10 μmol glucose unit/g wet wt., suggesting the release of lactate from muscle to hemolymph or carapace space (Jackson et al., 2001). The lactate increase during hypoxia was almost the same level as that in this species stored for 24 h in sawdust at 17 °C (Paterson, 1993b) but far lower than that in other crustaceans under environmental anoxia (Zebe, 1982; Gäde, 1984). In terms of free amino acids, only D- and L-alanine increased significantly from 12.5 to 18.9 μmol/g wet wt. in muscle during hypoxia and the D-alanine percentage remained at approximately 50%. This increase, however, was far lower than that in crayfish under hypoxia (Fujimori and Abe, 2002). Thus, an alternative anaerobic pathway, if any, might not work effectively in M. japonicus during hypoxia as was shown in other crustaceans (Zebe, 1982; Gäde, 1984). During recovery from hypoxia, phosphoarginine and ATP were replenished within 4 h. Lactate also returned to the control level within 2 h. In the crayfish O. limosus, the lactate level elevated during the initial period of recovery after anoxia and took over 24 h to return to the control level (Gäde, 1984). Since glycogen was largely mobilized during recovery of M. japonicus, the replenishment of phosphoarginine and ATP may be covered by the aerobic metabolism of glycogen without accumulation of lactate. These biochemical responses to hypoxia and recovery in M. japonicus were in agreement with those of functional anaerobiosis during swimming and recovery of other crustaceans except for the lactate production and clearance (Onnen and Zebe, 1983; Gäde, 1984). Therefore, these hypoxia responses of M. japonicus are predicted to occur also during locomotion by extensive tail flips. Several proteins in the muscle of M. japonicus were up- or down-regulated during hypoxia for 6 h (Fig. 4). Fructose bisphosphate aldolase, one of the major proteins in glycolytic pathway, was largely down-regulated during 6-h hypoxia (Fig. 4). This suggests the suppression of glycolysis in hypoxic muscle of M. japonicus and coincides with the data showing that decreases in glycogen are restrained during hypoxia (Fig. 1B). The three up-regulated proteins (2, 3, and 4 in Fig. 4) were all assigned to arginine kinase. They showed almost the same molecular mass of about 40 kDa but different isoelectric points. Invertebrate arginine kinase has been well characterized so far in mollusks, crustaceans, coelenterates, and echinoderms (Suzuki et al., 1997). Most crustacean arginine kinases have been identified as having almost the same molecular mass of ∼40 kDa consisting of a single polypeptide chain (Suzuki and Furukohri, 1994). The molecular mass coincides with that of the three proteins found in the present experiment (Fig. 4). The observation that three different protein spots gave the same enzyme protein cannot be accounted for in the present study. However, several isoforms have been reported for invertebrate arginine kinases, as well as vertebrate creatine kinases, with cytosolic and mitochondrial forms identified in muscle (Suzuki
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et al., 1997). Moreover, several invertebrates have also been reported to have three or more genes of arginine kinase (Uda et al., 2006). Thus, the three proteins are possibly isoforms of arginine kinase of M. japonicus. Arginine kinases from various invertebrates show high homology among invertebrate species as seen in Fig. 5, and are also homologous to creatine kinases of vertebrates and other phosphagen kinases of invertebrates. These kinases constitute a phosphagen (guanidino) kinase family (Suzuki and Furukohri, 1994). However, the fact that the two sequences, 2b and 4b in Fig. 5, were not matched completely with the known sequences of invertebrate arginine kinases and the same sequences, 2a and 4d, and 3b and 4a, were obtained from three different proteins may support the hypothesis that another isoform other than cytosolic and mitochondrial forms exists in the muscle of M. japonicus. This remains to be elucidated by gene cloning of these three proteins. The up-regulation of arginine kinase under hypoxia may represent a provision for oxygen recovery after a short period of hypoxia in the natural habitat of M. japonicus. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia. In conclusion, M. japonicus has large stores of fuel sources in the form of phosphoarginine and glycogen, which are used mainly under hypoxia and recovery, respectively. The phosphoarginine store effectively works as an ATP buffer during the periodic hypoxia that occurs in the natural habitat of this species, and the store is promptly recovered during oxygen supply as a buffer for the next hypoxic water. This is realized by the up-regulation of arginine kinase protein during anaerobiosis. Acknowledgement This work was partly supported by the Fisheries Agency of the Ministry of Agriculture, Forestry, and Fisheries of Japan. References Brouwer, M., Larkin, P., Brown-Peterson, N., King, C., Manning, S., Denslow, N., 2004. Effect of hypoxia on gene and protein expression in the blue crab, Callinectes sapidus. Mar. Environ. Res. 58, 787–792. Chen, J.-C., Chen, J.-S., 1998. Acid–base balance, ammonia, and lactate levels in the haemolymph of Penaeus japonicus during aerial exposure. Comp. Biochem. Physiol. A 121, 257–262. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K., 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102–1106. Fujimori, T., Abe, H., 2002. Physiological roles of free D- and L-alanine in the crayfish Procambarus clarkii with special reference to osmotic and anoxic stress responses. Comp. Biochem. Physiol. A 131, 893–900. Furusho, S., Umezaki, Y., Ishida, K., Honda, A., 1988. Changes in the concentration of ATP-related compounds and lactic acid in muscle of live prawn Penaeus japonicus during storage in sawdust. Nippon Suisan Gakkaishi 54, 1209–1212. Gäde, G., 1984. Effects of oxygen deprivation during anoxia and muscular work on the energy metabolism of the crayfish, Orconectes limosus. Comp. Biochem. Physiol. A 77, 495–502. Gorr, T.A., Cahn, J.D., Yamagata, H., Bunn, H.F., 2004. Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxiainducible factor-dependent. J. Biol. Chem. 279, 36038–36047.
Hervant, F., Garin, D., Mathieu, J., Freminet, A., 1999. Lactate metabolism and glucose turnover in the subterranean crustacean Niphargus virei during posthypoxic recovery. J. Exp. Biol. 202, 579–592. Hill, A.D., Taylor, A.C., Strang, R.H.C., 1991. Physiological and metabolic responses of the shore crab Carcinus maenas (L.) during environmental anoxia and subsequent recovery. J. Exp. Mar. Biol. Ecol. 150, 31–50. Hochachka, P.W., 1980. Living Without Oxygen: Closed and Open Systems in Hypoxia Tolerance. Harvard University Press, Cambridge, MA. Hochachka, P.W., Somero, G.N., 1984. Biochemical Adaptation. Princeton University Press, Princeton, NJ. Hochachka, P.W., Somero, G.N., 2002. Biochemical Adaptation — Mechanism and Process in Physiological Evolution. Oxford University Press, New York, NY. Jackson, D.C., Wang, T., Koldkjaer, P., Taylor, E.W., 2001. Lactate sequestration in the carapace of the crayfish Austropotamobius pallipes during exposure in air. J. Exp. Biol. 204, 941–946. Keppler, D., Decker, K., 1984. Glycogen. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 6. Verlag Chemie GmbH, Weinheim, pp. 11–17. Noll, F., 1984. L-(+)-Lactate. In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 6. Verlag Chemie GmbH, Weinheim, pp. 582–587. Ocampo, L., Patiño, D., Ramírez, C., 2003. Effect of temperature on hemolymph lactate and glucose concentrations in spiny lobster Panulirus interruptus during progressive hypoxia. J. Exp. Mar. Biol. Ecol. 296, 71–77. O'Farrell, P.H., 1975. High-resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021. Okuma, E., Abe, H., 1994a. Total D-amino and other free amino acids increase in the muscle of crayfish during seawater acclimation. Comp. Biochem. Physiol. A 109, 191–197. Okuma, E., Abe, H., 1994b. Simultaneous determination of D- and L-amino acids in the nervous tissues of crustaceans using precolumn derivatization with (+)-1-(9-fluorenyl)ethyl chloroformate and reversed-phase ion-pair high-performance liquid chromatography. J. Chromatogr. B 600, 243–250. Onnen, T., Zebe, E., 1983. Energy metabolism in the tail muscles of the shrimp Crangon crangon during work and subsequent recovery. Comp. Biochem. Physiol. A 74, 833–838. Paterson, B.D., 1993a. Respiration rate of the kuruma prawn, Penaeus japonicus Bate, is not increased by handling at low temperature (12 °C). Aquaculture 114, 229–235. Paterson, B.D., 1993b. The rise in inosine monophosphate and L-lactate concentrations in muscle of live penaeid prawns (Penaeus japonicus, Penaeus monodon) stressed by storage out of water. Comp. Biochem. Physiol. B 106, 395–400. Renaud, M.L., 1986. Detecting and avoiding oxygen deficient sea water by brown shrimp, Penaeus aztecus (Ives), and white shrimp Penaeus setiferus (Linnaeus). J. Exp. Mar. Biol. Ecol. 98, 283–292. Schmitt, A.S.C., Uglow, R.F., 1998. Metabolic responses of Nephrops norvegicus to progressive hypoxia. Aquat. Living Resour. 11, 87–92. Strong, S.J., Ellington, W.R., 1995. Isolation and sequence analysis of the gene for arginine kinase from the chelicerate arthropod, Limulus polyphemus: insights into catalytically important residues. Biochim. Biophys. Acta 1246, 197–200. Suzuki, T., Furukohri, T., 1994. Evolution of phosphagen kinase: primary structure of glycocyamine kinase and arginine kinase from invertebrates. J. Mol. Biol. 237, 353–357. Suzuki, T., Kawasaki, Y., Furukohri, T., 1997. Evolution of phosphagen kinase: isolation, characterization and cDNA-derived amino acid sequence of twodomain arginine kinase from the sea anemone Anthopleura japonicus. Biochem. J. 328, 301–306. Suzuki, T., Fukuta, H., Nagato, H., Umekawa, M., 2000. Arginine kinase from Nautilus pompilius, a living fossil. J. Biol. Chem. 275, 23884–23890. Uda, K., Fujimoto, N., Akiyama, Y., Mizuta, K., Tanaka, K., Ellington, W.R., Suzuki, T., 2006. Evolution of the arginine kinase gene family. Comp. Biochem. Physiol. D 1, 209–218. Willsie, J.K., Clegg, J.S., 2001. Nuclear p26, a small heat shock/α-crystallin protein, and its relationship to stress resistance in Artemia franciscana embryos. J. Exp. Biol. 204, 2339–2350. Zebe, E., 1982. Anaerobic metabolism in Upogebia pugettensis and Callianassa californiensis (Crustacea, Thalassinidea). Comp. Biochem. Physiol. B 72, 613–617.
Metabolic responses and arginine kinase expression under hypoxic stress of the kuruma prawn Marsupenaeus japonicus Hiroki Abe ⁎, Shun Hirai, Shigeru Okada Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo, Tokyo 113-8657, Japan Received 4 April 2006; received in revised form 10 August 2006; accepted 18 August 2006 Available online 30 August 2006
Abstract In response to hypoxia at PO2 1.3–1.7 mg/L for 6 h, the kuruma prawn Marsupenaeus (Penaeus) japonicus showed a dramatic decrease in phosphoarginine storage in muscle, with normal levels restored during 4-h post-hypoxic recovery. Large stores of muscle glycogen only decreased between 4 and 6 h during hypoxia, but greatly diminished during recovery. Muscle ATP levels and energy charge decreased only slightly under hypoxia. Lactate levels increased slightly during hypoxia and promptly returned to control levels during recovery. These data indicate that phosphoarginine works in muscle as an ATP buffer during hypoxia and glycogen is utilized as an energy source during recovery. Under hypoxia, up- and down-regulated proteins were identified after 2D electrophoresis and partial sequences were obtained after protease digestion. Fructose bisphosphate aldolase was down-regulated during hypoxia, suggesting the suppression of glycolysis under hypoxia. Several partial sequences from three protein spots up-regulated under hypoxia were all assigned to arginine kinase, suggesting the existence of several isoforms of arginine kinase in the muscle of M. japonicus. This arginine kinase up-regulation under hypoxia may indicate a provision for oxygen re-supply after anaerobiosis. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia. © 2006 Elsevier Inc. All rights reserved. Keywords: Arginine kinase; Phosphoarginine; ATP; Energy metabolism; Glycogen; Hypoxia; Kuruma prawn; Marsupenaeus japonicus
1. Introduction Aquatic invertebrates encounter periodic environmental hypoxia or anoxia and evolve effective anaerobic mechanisms to cope with reduced ambient oxygen concentrations (Hochachka, 1980; Hochachka and Somero, 1984). In contrast to well-known hypoxia-tolerant invertebrates such as bivalve mollusks and annelids, hypoxia tolerance appears to be highly species-specific in crustaceans (Schmitt and Uglow, 1998). In decapod crustaceans, burrowing species usually show higher hypoxia tolerance than free-swimming species. The burrowing and hypoxia-tolerant decapods often maintain large stores of energy sources such as glycogen and phosphoarginine and reduce their metabolic rate under hypoxia (Hervant et al., 1999). During anaerobiosis, crustaceans have been considered to employ only classical anaerobic glycolysis, leading to L-lactate as the sole end⁎ Corresponding author. Tel.: +81 3 5841 5296; fax: +81 3 5841 8166. E-mail address: [email protected] (H. Abe). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.08.027
product, whereas in some species alanine and succinate have also been reported to be accumulated (Zebe, 1982; Onnen and Zebe, 1983; Gäde, 1984; Hill et al., 1991). We also confirmed that the crayfish Procambarus clarkii accumulated D- and L-alanine besides L-lactate in muscle and hepatopancreas under severe hypoxia below 0.1 mg O2/L (Fujimori and Abe, 2002). Under severe environmental hypoxia, crustaceans need to regulate the production of some key enzyme proteins to survive in harsh environments, as is known in other animals (Hochachka and Somero, 2002). There have been only a few reports so far on gene and protein expression in crustaceans during hypoxia: heat shock/α-crystallin protein, p26, in Artemia franciscana embryo (Willsie and Clegg, 2001); heat induced factor mediated globin gene in Daphnia magna (Gorr et al., 2004); and several genes including Mn-superoxide dismutase gene and hemocyanin protein in blue crab Callinectes sapidus (Brouwer et al., 2004). It is of particular interest to clarify the regulation of gene and protein expression levels in relation to energy metabolism under oxygen-limiting conditions.
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The kuruma prawn Marsupenaeus (Penaeus) japonicus is a swimming and burrowing species and the most commercially valuable penaeid species that is widely cultured in Japan and other Asian countries. The prawn is transported live to markets and consumers in dry sawdust at low temperature (just above 10 °C) and is kept alive for several days. There are a few reports in the literature on the hypoxia tolerance of this species during aerial exposure (Furusho et al., 1988; Paterson, 1993a,b; Chen and Chen, 1998), but no report on the metabolic responses of M. japonicus in relation to protein over-expression during hypoxia. The objective of this study was to obtain information on the metabolic responses of M. japonicus under hypoxic stress and during post-hypoxic recovery, and on up- and down-regulated proteins during hypoxia. 2. Materials and methods 2.1. Animals and hypoxia stress loading Live adult M. japonicus weighing approximately 20 g were obtained from a culture farm in Miyazaki Prefecture, Japan and transported live by air to the laboratory in dry sawdust at low temperature. They were reared in a 60-L aquarium supplied with aerated circulating seawater (filtrated natural seawater of 35 ppt) and maintained at 15 °C. At the bottom of the aquarium was a fine sand layer of 10 cm in depth. The animals were fed daily with dry pellets for prawns, but were not fed for 2 days prior to the experiments. After acclimation to the aquarium conditions, the animals were exposed to hypoxia (PO2 = 1.3–1.7 mg/L, ∼3 kPa) for 6 h under constant bubbling of nitrogen gas. The water surface was covered with a plastic sheet during hypoxia and the sand layer was removed. After hypoxia, the animals were recovered for 4 h in hyperoxic conditions with vigorous aeration. During hypoxia and recovery, 4 to 5 individuals were taken out at 2-h intervals. Special care was taken to avoid tail flips by prawn during sampling. After decapitation, the tail muscle and hepatopancreas were excised and quickly frozen in liquid nitrogen. A perchloric acid extract was prepared from a 1-g sample as previously described (Okuma and Abe, 1994b). All prawns used in the experiment were in the intermolt stage. 2.2. Analytical methods Free amino acid analysis was performed on an amino acid autoanalyzer (L-8500A; Hitachi, Tokyo, Japan). Phosphoarginine was determined by high-performance liquid chromatography (HPLC). The HPLC system (Jasco, Tokyo, Japan) consisted of a PU-1580 pump, a DG-1580-53 line degasser, an LG-158002 ternary gradient unit, a CO-1565 column oven, an AS-2057 autosampler with a cooling system, a UV-1570 UV–VIS detector, and an 807-IT integrator. An anion-exchange column (Shim-pack CLC-NH2; Shimadzu, Kyoto, Japan) was used with a guard column. Elution was conducted isocratically with 10 mM sodium phosphate buffer, pH 2.6, containing 20% acetonitrile at a flow rate of 1 mL/min at 40 °C. Phosphoarginine was detected at 205 nm.
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Nucleotides were determined on the above HPLC system essentially according to the method of Okuma and Abe (1994a). Muscle glycogen and L-lactate were determined enzymatically according to the methods of Keppler and Decker (1984) and Noll (1984), respectively. All chemicals were of analytical grade and were obtained from Sigma-Aldrich (St. Louis, MO, USA) or Wako Pure Chemical Industries (Osaka, Japan) unless otherwise stated. 2.3. Protein extraction for two-dimensional (2D) electrophoresis Muscle tissue was homogenized with a Polytron homogenizer (Kinematica, Littau-Lucerne, Switzerland) in 4 volumes of 50 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and centrifuged at 10 000 ×g for 30 min. Proteins in the supernatant were salted out with ammonium sulfate between 30% and 70% saturation. The precipitate after centrifugation as above was dissolved in 5 mM potassium phosphate buffer, pH 7.4, containing 1 mM EDTA and 0.2 mM PMSF and dialyzed overnight against the same buffer. After centrifugation as above, the supernatant obtained was subjected to 2D electrophoresis. Protein concentration was determined with a protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). 2.4. Analytical 2D electrophoresis and peptide mapping with limited proteolysis Analytical 2D electrophoresis was performed using the method of O'Farrell (1975). The first dimensional isoelectric focusing was conducted using 4% polyacrylamide gel containing 8 M urea, 2% Triton X-100, and 1.6% and 0.4% Ampholine (Pharmacia, Uppsala, Sweden), pH 5–8 and 3–10, respectively. The second dimensional sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) was performed on an 11.5% slab gel. After electrophoreses, the gel was stained with 0.25% Coomassie brilliant blue R-250. Electrophoretic patterns were analyzed with an ImageMaster VDS-CL (Amersham Bioscience, Cleveland, OH, USA). After 2D electrophoresis, proteins up- or down-regulated after 6-h hypoxia were cut out from the gel and equilibrated in 0.125 M Tris–HCl, pH 6.8, containing 0.1% SDS for 30 min with shaking. The electrophoresis was repeated over 10 times and the protein spots of interest were pooled and concentrated. Each protein was subjected to limited proteolysis with Staphylococcus aureus V8 protease (Wako) during SDS-PAGE according to the method of Cleveland et al. (1977). 2.5. Determination of amino acid sequence of peptide fragments The proteins on Coomassie-stained SDS-PAGE gels were electrically transferred onto Immobilon polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA) and subjected to a protein sequencer (Applied Biosystem model 492HT; Foster City, CA, USA). The amino acid sequences of peptide fragments were compared using a BLAST search.
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Fig. 1. Changes in phosphoarginine (A) and glycogen (B) levels in the muscle and hepatopancreas of the kuruma prawn M. japonicus in 35 ppt seawater at 15 °C during hypoxia at PO2 1.3–1.7 mg/L (around 3 kPa) for 6 h and subsequent recovery for 4 h. ⁎P b 0.05, ⁎⁎P b 0.01, ⁎⁎⁎P b 0.001 compared with 0 time control (n = 4–5).
2.6. Statistical analyses The results of metabolite analyses are presented as mean ± S.D. (n = 4–5). The data were analyzed statistically using one-way ANOVA followed by post-hoc comparison of means (LSD test). In all comparison, 0 time value was used as control. 3. Results 3.1. Biochemical responses to hypoxia and recovery Under hypoxia at PO2 values between 1.3 and 1.7 mg/L (approx. 3 kPa) for 6 h, only three animals died between 4 and 6 h and the survival rate was 90%. The large store of
phosphoarginine accumulated in the muscle of control prawn (31 μmol/g wet wt., n = 5) decreased dramatically to one-fifth (6.1 μmol/g wet wt., n = 4–5) during hypoxia and increased linearly to the control level after 4 h of recovery (Fig. 1A). This was also the case in hepatopancreas, where the concentration of phosphoarginine was far lower than that in muscle. Muscle glycogen levels decreased during the last 2 h of hypoxia and the decrease was significant statistically (Fig. 1B). A much greater reduction in muscle glycogen level was found during recovery. In hepatopancreas, the glycogen level was only half of that in muscle and decreased significantly during the last 2 h of hypoxia. In contrast to muscle, glycogen levels in hepatopancreas increased to the control level during recovery.
Fig. 2. Changes in nucleotide levels in the muscle (A) and hepatopancreas (B) of M. japonicus during hypoxia and recovery. Refer to the legend of Fig. 1 for experimental conditions and significances.
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increased significantly and stoichiometrically with the ATP decrease during hypoxia (Fig. 2B). Under hypoxia, L-lactate increased in muscle and returned to the control level after 2 h of recovery (Fig. 3). However, the increase in lactate during hypoxia (2.3 μmol/g wet wt.) was far lower than the decrease in glycogen (10 μmol glucose unit/g wet wt.) in muscle (Fig. 1B). Of all amino acid determined, only D- and L-alanine increased significantly from control level (12.5 μmol/g) to 18.9 μmol/g during 6-h hypoxia (data not shown). 3.2. Changes in protein expression under hypoxic stress
Fig. 3. Changes in L-lactate in the muscle of M. japonicus during hypoxia and recovery. Refer to the legend of Fig. 1 for experimental conditions and significances.
Fig. 2 shows the changes in nucleotides during hypoxia and recovery. Muscle ATP levels decreased moderately during hypoxia and then increased back to the control level during recovery. As a result, the adenylate energy charge was maintained between 0.84 and 0.97 in muscle during hypoxia and recovery. The lower ATP levels in hepatopancreas also decreased during hypoxia and returned to the control level during recovery. In hepatopancreas, however, the energy charge was as low as 0.7 even in control animals, and declined to 0.5 during hypoxia. Along with the decrease in ATP in hepatopancreas, AMP levels
Fig. 4 shows the 2D electrophoretic profiles of muscle protein solutions from control and 6-h hypoxic individuals of M. japonicus (250 μg each). Many of the protein spots were upor down-regulated under hypoxia, but four spots (nos. 1–4) were selected in the present experiment based on the reproducibility and differences of the spots between control and hypoxic individuals (Fig. 4C–F). From protein spot 1, for which expression decreased to onefifth under hypoxia compared with control, three partial amino acid sequences (1a–c) were obtained (Table 1). Of these sequences, 1b was assigned to fructose bisphosphate aldolase (EC 4.1.2.13) of crucian carp Carassius auratus, mouse Mus musculus, and human Homo sapiens using a BLAST search. Sequence 1b showed 87% amino acid identity to these aldolases. Sequences, 1a and 1c, showed no matches with the BLAST database. All other partial amino acid sequences from proteins, 2, 3, and 4, of which expressions increased 2- to 5-fold under hypoxia, were assigned to arginine kinases (EC 2.7.3.3)
Fig. 4. Two-dimensional electrophoretic profiles of proteins from the muscle of control M. japonicus (A) and that exposed to 6-h hypoxia (B). Arrows with numbers indicate the protein spots down- or up-regulated under hypoxia compared with the control. Panels C and D and those E and F are magnified photos in the related regions of panels A and B, respectively.
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Table 1 Partial amino acid sequences of peptides obtained from the protein spots 1–4 in Fig. 4 which were down- or up-regulated under hypoxic stress of M. japonicus Peptide no.
Partial amino acid sequence
1a 1b
NAKKYRQLKFTADKAFR LSQRYAQYKKDGADFAIKKYDDL
1c 2a
STTQGLDDLGQQQGAAILPNFI AYTLFAPLFDPIIED
2b 3a 3b
DYAVGFKQTDKKPNK AELKGTYFPKTYMSKGQTD VQQKLIDDHFLFKEG
4a 4b
VQQKLIDDHFLFKEG AQYKEMETKVSSTLSMEVSSEL
4c 4d
KELKGLYFKVSGMKAQYETALG AYTLFAPLFDPIIED
Homologous proteins (sources)
Identity (%)
Fructose bisphosphate aldolase (Carassius auratus, AAA84887) Fructose bisphosphate aldolase (Mus musculus, AAA37210) Fructose bisphosphate aldolase (Homo sapiens, CAA30979)
87 87 87
Arginine Arginine Arginine Arginine
100 100 100 86
kinase (Marsupenaeus japonicus, P51545) kinase (Pachygrapsus marmoratus, AAG01175) kinase (Callinectes sapidus, AAF43436) kinase (Marsupenaeus japonicus)
Arginine kinase (Marsupenaeus japonicus) Arginine kinase (Pachygrapsus marmoratus) Arginine kinase (Eriocheir sinensis, AAF43437) Same as 3b Arginine kinase (Drosophila melanogaster, P48610) Arginine kinase (Eriocheir sinensis) Arginine kinase (Carcinus maenas, AAD48470)
100 100 100 93 93 93
Same as 2a
Homologous proteins were obtained from the BLAST database. GenBank accession numbers are shown after each species name.
from invertebrates, including M. japonicus, with high amino acid identities of 86–100% (Table 1). All of these invertebrates are crustaceans, except for fruit fly Drosophila melanogaster, and all are crab species, except for M. japonicus. Several partial sequences obtained from different proteins showed the same
sequences, as observed for 2a and 4d, and 3b and 4a. On the other hand, 3a and 4c matched with no protein sequences. Fig. 5 shows the amino acid sequences of arginine kinase from M. japonicus and three crab species, as well as partial sequences obtained in the present experiment. The amino acid
Fig. 5. Alignment of deduced amino acid sequences of arginine kinases from representative crustaceans with partial sequences (in bold letters) identified in the muscle of M. japonicus under hypoxia. Asterisks indicate the identical amino acid residues among the four sequences. In parentheses, sequence numbers shown in Table 1 are indicated. Residues interacting with the amino and carboxyl groups of substrate arginine are boxed. Aspartate and arginine residues, key residues in arginine kinase function, are boxed by dotted line (Suzuki et al., 2000). Refer to Table 1 for accession number for each sequence.
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sequences of crustacean arginine kinase show high homology and four amino acid residues interacting with the substrate arginine are conserved in all crustacean enzymes. Two additional residues, aspartate and arginine, are also conserved and are supposed to be essential for enzyme function (Strong and Ellington, 1995; Suzuki et al., 2000). In the partial sequences obtained in the present experiment, sequences 2a (4d) and 3b (4a) coincided completely with those of crustacean arginine kinases. Other partial sequences, 2b and 4b, were also similar to the crustacean sequences, but showed several amino acid substitutions. 4. Discussion When M. japonicus was subjected to severe hypoxia below 0.1 mg O2/L under bubbling of nitrogen gas, the survival rate was extremely low even during 3-h hypoxia (data not shown). Thus, M. japonicus is more oxygen-sensitive than crayfish P. clarkii which easily survives for at least 12 h under the same conditions (Fujimori and Abe, 2002). Based on these results, PO2 was set at approximately 3 kPa in the present study. This PO2 level may occur in shallow coastal regions during summer and was known to be avoided by some Penaeus prawns (Renaud, 1986). The muscle phosphoarginine concentration was extremely high, 31 μmol/g, in normoxic M. japonicus and dramatically decreased to 6.1 μmol/g during hypoxia (Fig. 1A). This phosphoarginine level was 1.5-fold higher than that in crayfish Orconectes limosus (Gäde, 1984). In contrast, muscle ATP levels only moderately decreased under hypoxia (Fig. 2A). These data suggest that the ATP level in muscle was effectively buffered by phosphoarginine. The muscle glycogen level decreased only between 4 and 6 h during hypoxia (Fig. 1B). Thus, in M. japonicus under hypoxia of ∼3 kPa for 6 h, muscle energy may be supplied mainly by phosphoarginine, followed by glycogenolysis. The level of muscle glycogen was almost the same as that in crayfish O. limosus (Gäde, 1984) and P. clarkii (Fujimori and Abe, 2002). Muscle glycogen decreased only during the last 2 h of hypoxia and largely during recovery (Fig. 1B). This suggests that unlike P. clarkii, M. japonicus mainly depends on glycogenolysis during recovery from hypoxia, which leads to prompt recovery of phosphoarginine and ATP levels (Figs. 1A and 2A). No increase in glucose was found throughout hypoxia and recovery periods (data not shown). This is consistent with the case of spiny lobster Panulirus interruptus under hypoxia (Ocampo et al., 2003). The muscle ATP level was almost the same as that reported previously in this species (Furusho et al., 1988; Paterson, 1993b) but the decrease of ATP during hypoxia was far lower than that during aerial exposure in dry sawdust in these reports, although the experimental period was longer in aerial exposure (24 h) than hypoxia in the present study (6 h). These data may suggest that some different anaerobic mechanisms work between aerial exposure and environmental hypoxia. In hepatopancreas, phosphoarginine and glycogen levels were far lower than in muscle and decreased during hypoxia, suggesting that ATP buffering by phosphoarginine is not effective in hepatopancreas. The content of ATP and energy
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charge were also very low in hepatopancreas and both lowered during hypoxia. Thus, the energy state and utilization of hepatopancreas in normoxic and hypoxic water may be quite different from those of muscle. Under hypoxia, the increase in muscle lactate was limited to 2.3 μmol/g wet wt., whereas the decrease in glycogen was 10 μmol glucose unit/g wet wt., suggesting the release of lactate from muscle to hemolymph or carapace space (Jackson et al., 2001). The lactate increase during hypoxia was almost the same level as that in this species stored for 24 h in sawdust at 17 °C (Paterson, 1993b) but far lower than that in other crustaceans under environmental anoxia (Zebe, 1982; Gäde, 1984). In terms of free amino acids, only D- and L-alanine increased significantly from 12.5 to 18.9 μmol/g wet wt. in muscle during hypoxia and the D-alanine percentage remained at approximately 50%. This increase, however, was far lower than that in crayfish under hypoxia (Fujimori and Abe, 2002). Thus, an alternative anaerobic pathway, if any, might not work effectively in M. japonicus during hypoxia as was shown in other crustaceans (Zebe, 1982; Gäde, 1984). During recovery from hypoxia, phosphoarginine and ATP were replenished within 4 h. Lactate also returned to the control level within 2 h. In the crayfish O. limosus, the lactate level elevated during the initial period of recovery after anoxia and took over 24 h to return to the control level (Gäde, 1984). Since glycogen was largely mobilized during recovery of M. japonicus, the replenishment of phosphoarginine and ATP may be covered by the aerobic metabolism of glycogen without accumulation of lactate. These biochemical responses to hypoxia and recovery in M. japonicus were in agreement with those of functional anaerobiosis during swimming and recovery of other crustaceans except for the lactate production and clearance (Onnen and Zebe, 1983; Gäde, 1984). Therefore, these hypoxia responses of M. japonicus are predicted to occur also during locomotion by extensive tail flips. Several proteins in the muscle of M. japonicus were up- or down-regulated during hypoxia for 6 h (Fig. 4). Fructose bisphosphate aldolase, one of the major proteins in glycolytic pathway, was largely down-regulated during 6-h hypoxia (Fig. 4). This suggests the suppression of glycolysis in hypoxic muscle of M. japonicus and coincides with the data showing that decreases in glycogen are restrained during hypoxia (Fig. 1B). The three up-regulated proteins (2, 3, and 4 in Fig. 4) were all assigned to arginine kinase. They showed almost the same molecular mass of about 40 kDa but different isoelectric points. Invertebrate arginine kinase has been well characterized so far in mollusks, crustaceans, coelenterates, and echinoderms (Suzuki et al., 1997). Most crustacean arginine kinases have been identified as having almost the same molecular mass of ∼40 kDa consisting of a single polypeptide chain (Suzuki and Furukohri, 1994). The molecular mass coincides with that of the three proteins found in the present experiment (Fig. 4). The observation that three different protein spots gave the same enzyme protein cannot be accounted for in the present study. However, several isoforms have been reported for invertebrate arginine kinases, as well as vertebrate creatine kinases, with cytosolic and mitochondrial forms identified in muscle (Suzuki
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et al., 1997). Moreover, several invertebrates have also been reported to have three or more genes of arginine kinase (Uda et al., 2006). Thus, the three proteins are possibly isoforms of arginine kinase of M. japonicus. Arginine kinases from various invertebrates show high homology among invertebrate species as seen in Fig. 5, and are also homologous to creatine kinases of vertebrates and other phosphagen kinases of invertebrates. These kinases constitute a phosphagen (guanidino) kinase family (Suzuki and Furukohri, 1994). However, the fact that the two sequences, 2b and 4b in Fig. 5, were not matched completely with the known sequences of invertebrate arginine kinases and the same sequences, 2a and 4d, and 3b and 4a, were obtained from three different proteins may support the hypothesis that another isoform other than cytosolic and mitochondrial forms exists in the muscle of M. japonicus. This remains to be elucidated by gene cloning of these three proteins. The up-regulation of arginine kinase under hypoxia may represent a provision for oxygen recovery after a short period of hypoxia in the natural habitat of M. japonicus. This is consistent with the prompt replenishment of phosphoarginine stores during recovery from hypoxia. In conclusion, M. japonicus has large stores of fuel sources in the form of phosphoarginine and glycogen, which are used mainly under hypoxia and recovery, respectively. The phosphoarginine store effectively works as an ATP buffer during the periodic hypoxia that occurs in the natural habitat of this species, and the store is promptly recovered during oxygen supply as a buffer for the next hypoxic water. This is realized by the up-regulation of arginine kinase protein during anaerobiosis. Acknowledgement This work was partly supported by the Fisheries Agency of the Ministry of Agriculture, Forestry, and Fisheries of Japan. References Brouwer, M., Larkin, P., Brown-Peterson, N., King, C., Manning, S., Denslow, N., 2004. Effect of hypoxia on gene and protein expression in the blue crab, Callinectes sapidus. Mar. Environ. Res. 58, 787–792. Chen, J.-C., Chen, J.-S., 1998. Acid–base balance, ammonia, and lactate levels in the haemolymph of Penaeus japonicus during aerial exposure. Comp. Biochem. Physiol. A 121, 257–262. Cleveland, D.W., Fischer, S.G., Kirschner, M.W., Laemmli, U.K., 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252, 1102–1106. Fujimori, T., Abe, H., 2002. Physiological roles of free D- and L-alanine in the crayfish Procambarus clarkii with special reference to osmotic and anoxic stress responses. Comp. Biochem. Physiol. A 131, 893–900. Furusho, S., Umezaki, Y., Ishida, K., Honda, A., 1988. Changes in the concentration of ATP-related compounds and lactic acid in muscle of live prawn Penaeus japonicus during storage in sawdust. Nippon Suisan Gakkaishi 54, 1209–1212. Gäde, G., 1984. Effects of oxygen deprivation during anoxia and muscular work on the energy metabolism of the crayfish, Orconectes limosus. Comp. Biochem. Physiol. A 77, 495–502. Gorr, T.A., Cahn, J.D., Yamagata, H., Bunn, H.F., 2004. Hypoxia-induced synthesis of hemoglobin in the crustacean Daphnia magna is hypoxiainducible factor-dependent. J. Biol. Chem. 279, 36038–36047.
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