Physiological roles of free D- and L-alanine in the crayfish Procambarus clarkii with special reference to osmotic and anoxic stress responses

Under hyper-salinity stress from freshwater to 17 and 25 ppt seawater, red swamp crayfish Procambarus clarkii largely accumulated D- and L-alanine together with glycine, L-glutamine, and L-proline in both muscle and hepatopancreas. The increases of D- and L-alanine in muscle were the highest in all amino acids and reached 6.8- and 5.4-fold, respectively, from freshwater to 25 ppt seawater. These results indicate that both D- and L-alanine are the most potent osmolytes for intracellular isosmotic regulation in crayfish as well as other crustaceans thus far examined. Under anoxia stress below 0.1 mg/l dissolved oxygen for 12 h and subsequent recovery in normoxia for 12 h in freshwater, 17 and 25 ppt seawater, muscle ATP decreased dramatically in all salinity levels and almost depleted in seawater. Along with the decrease of muscle glycogen level, the significant increase of L-lactate was found in muscle, hepatopancreas, and hemolymph for each salinity level, suggesting the transport of L-lactate from muscle into hepatopancreas via hemolymph. Under anoxia, D- and L-alanine also largely increased in both muscle and hepatopancreas for each salinity level. The increase was much higher in seawater than in freshwater. Thus, both D- and L-alanine are possible to be anaerobic end products during prolonged anaerobiosis of this species.     

Total D-amino and other free amino acids increase in the muscle of crayfish during seawater acclimation

Freshwater crayfish contained smaller amounts of total free amino acids in the muscle than marine prawns and lobsters. In the muscle of seawater acclimated crayfish, however, total amino acids were elevated by 3.7-fold and reached almost equivalent values to those commonly found in marine species. Glycine, alanine, proline, asparagine, and glutamine were the major amino acids elevated during seawater acclimation. Changes in alanine were most pronounced with its concentration increasing by 112 mmol/kg water (15.2-fold approx.). Crayfish muscle contains significant amounts of D-amino acids (mostly D-alanine) and these rose from 1.85 to 58.5mmol/kg water during seawater acclimation. D-Alanine in the seawater crayfish corresponds to almost one half of the total (D + L) alanine pool.     

Occurrence of Alanine Racemase in Crustaceans and the Changes of the Properties During Seawater Acclimation of Crayfish

Alanine racemase activity was detected in the muscle and hepatopancreas of six macruran species. Optimal pH was around 8.5 irrespective of species and tissues. Apparent Michaelis constants ranged from 48 to 157 mM for muscle enzyme and 35 to 239 mM for hepatopancreas enzyme. The enzyme specifically catalyzed the racemization of d- and l-alanine. The enzyme did not require pyridoxal 5′-phosphate as well as FAD as a cofactor. Pyruvate and l-alanine showed strong inhibition in the direction of d to l.During seawater acclimation of crayfish Procambarus clarkii, alanine racemase activity increased about twice in muscle and 1.5 times in hepatopancreas. Michaelis constant, on the other hand, decreased 33% for muscle enzyme and 65% for hepatopancreas enzyme, suggesting the increase of substrate affinity during seawater acclimation. The activity in the physiological pH range (6.5–7.5) also increased with increasing salinity.     

Purification and properties of alanine racemase from crayfish Procambarus clarkii

Fresh water crayfish Procambarus clarkii is known to accumulate d-alanine remarkably in muscle after seawater acclimation, accompanied by an increase in alanine racemase activity. We have purified alanine racemase from crayfish muscle to homogeneity. The enzyme is a monomeric protein with a molecular mass of 58 kDa. It is highly specific to alanine and does not racemize l-serine, l-aspartate, l-glutamate, l-valine and l-arginine. The enzyme shows the highest activity at pH 9.0 in the conversion of l- to d-alanine and at pH 8.5 in the reverse conversion. Properties such as amino acid sequence, quaternary structure, pyridoxal 5′-phosphate (PLP)-dependency, pH-dependency and kinetic parameters seem to be distinct from those of the microbial alanine racemases. Various salts including NaCl at concentrations around seawater level were potently inhibitory for the activity in both of l- to -d and d- to -l direction.     

Purification and characterization of alanine racemase from hepatopancreas of black-tiger prawn, Penaeus monodon

Alanine racemase has been purified to homogeneity from the hepatopancreas of the black tiger prawn, Panaeus mondon. The enzyme depends on pyridoxal 5′-phosphate and consists of two subunits with an identical molecular weight of 41,000. V_max and K_m values for -alanine are 460 μmol/min/mg and 50 mM, and those for -alanine are 94 μmol/min/mg and 24 mM, respectively. The enzyme is highly specific toward alanine. Among other amino acids examined, only serine served as a substrate: -serine was racemized at a rate of approximately 0.5% of that of -alanine. The prawn enzyme is immunochemically distinguishable from the enzymes of Bacillus stearothermophilus and Schizosaccharomyces pombe, which resemble each other. The prawn enzyme is activated and stabilized by the presence of monovalent anions including chloride. This is consistent with the previous hypothesis (e.g. E. Fujita, E. Okuma, H. Abe, Comp. Biochem. Physiol. 116A (1997) 83–87) that -alanine serves as an osmoregulator in marine and euryhaline animals.     

C-type lectin from red swamp crayfish Procambarus clarkii participates in cellular immune response

Lectins are potential immune recognition proteins. In this study, a novel C-type lectin (Pc-Lec1) is reported in freshwater crayfish Procambarus clarkii. Pc-Lec1 encodes a protein of 163 amino acids with a putative signal peptide and a single carbohydrate recognition domain. It was constitutively expressed in various tissues of a normal crayfish, especially in the hepatopancreas and gills. Expressions of Pc-Lec1 were up-regulated in the hepatopancreas and gills of crayfish challenged with Vibrio anguillarum, Staphylococcus aureus, or the white spot syndrome virus. Recombinant mature Pc-Lec1 bound bacteria and polysaccharides (peptidoglycan, lipoteichoic acid, and lipopolysaccharide) but did not agglutinate bacteria. Pc-Lec1 enhanced hemocyte encapsulation of the sepharose beads in vitro, and the blocking of beads by a polyclonal antibody inhibited encapsulation. Pc-Lec1 promoted clearance of V. anguillarum in vivo. These results suggest that Pc-Lec1 is a pattern recognition receptor and participates in cellular immune response. Pc-Lec1 performs its function as an opsonin by enhancing the encapsulation or clearance of pathogenic bacteria.     

HPLC determination of the distribution of D-amino acids and effects of ecdysis on alanine racemase activity in kuruma prawn Marsupenaeus japonicus

The distribution of D-amino acids was examined on several tissues of kuruma prawn Marsupenaeus japonicus. D-Alanine was found in all tissues, and the ratio of D-alanine to total alanine ranged from 18.7 to 43.7% depending on the tissues. Of these tissues, muscle, heart, and gill contained a relatively large amount of D-alanine. Nervous tissue and eye, on the other hand, contained a large amount of D-aspartate. D-Glutamate was specifically detected in testis. The percentage of D-glutamate to total glutamate was over 50% in testis, suggesting the existence of the biosynthetic enzyme in this tissue. The changes of alanine racemase activity were determined in the muscle and hepatopancreas of M. japonicus before and after molting. The activity after molting increased twice in the muscle. On the other hand, it was not changed in the hepatopancreas. These data suggest that D-alanine plays an important role in the muscle during ecdysis. However, the free D-alanine level in the muscle was not changed significantly before and after ecdysis. From these data, several D-amino acids are considered to be utilized in some essential physiological phenomena in the different tissues of the prawn.     

The concentrations of antioxidant compounds in the hepatopancreas, the gills and muscle of some freshwater crayfish species

We examined the concentrations of vitamin E (vit E), sulphydryl groups (-SH), total protein and protein electrophoretic profiles in the hepatopancreas, the gills and muscle of three freshwater crayfish species: Noble crayfish (Astacus astacus), stone crayfish (Austropotamobius torrentium) and spiny cheek crayfish (Orconectes limosus). Vit E concentration in the hepatopancreas of O. limosus was lower compared to A. astacus, while in the gills of O. limosus it was lower compared to both A. astacus and A. torrentium. The concentration of -SH groups was lower in the hepatopancreas of A. astacus compared to A. torrentium and O. limosus. In the gills of A. astacus and A. torrentium the concentration of -SH groups was higher compared to O. limosus. Protein concentration was higher in the hepatopancreas of A. torrentium compared to A. astacus and O. limosus. A lower protein concentration in muscle of A. torrentium was found compared to O. limosus and A. astacus. Electrophoretic analysis of proteins indicated species and tissue specifities between investigated crayfish species. Our results represent the first study of its kind and provide the basis for future studies that will consider our reported parameters as potential biomarkers for biomonitoring of basic environmental conditions and some anthropogenic impacts.     

Mobilization of copper among tissues in the estuarine crab Scylla serrata (Forskal) under imposed starvation

The quantitative changes in copper free and bound to proteins in haemolymph and different forms of copper in muscle and hepatopancreas under imposed starvation were studied in the estuarine mud crab Scylla serrata. During the course of starvation, both haemolymph copper free and bound to proteins significantly declined and the regression analyses of these data further revealed that the haemolymph copper-free proteins were more affected than copper-bound proteins. The multiple stress condition namely injury and exsanguination along with starvation resulted in an earlier release and/or degradation of both these proteins. Hepatopancreas periodically accumulates and releases copper during starvation. The copper levels in haemolymph and hepatopancreas during different days of starvation showed a close inverse relationship between these two tissues. These changes in hepatopancreas were predominantly reflected in the copper that exists in association with low molecular weight substances. It is found that the copper thus accumulated was partly released back into haemolymph and a fraction may be excreted. This study also indicates the major role played by the low molecular weight substances in accommodation, detoxification and mobilization of copper in the decapod hepatopancreas during imposed starvation.     

Elongation factor 1Bgamma (eEF1Bgamma) expression during the molting cycle and cold acclimation in the crayfish Procambarus clarkii

Eukaryotic elongation factor 1Bγ (eEF1Bγ) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bγ has properties which suggest sensory or regulatory activities. We have cloned eEF1Bγ from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bγ and 65% identity to Artemia salina eEF1Bγ. We measured eEF1Bγ expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bγ expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bγ expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bγ was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bγ expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bγ were found after 1 week of acclimation to 4 °C. These results show that eEF1Bγ is regulated at the mRNA level with tissue-specific differences in expression patterns.     

Amino acids in haemolymph, single fibres and whole muscle from the claw of freshwater crayfish acclimated to different osmotic environments

The concentrations of free amino acids were measured in whole claw muscle, single fibres and haemolymph of Australian freshwater crayfish, Cherax destructor, during the intermoult stage. The average total pool of amino acids in short-sarcomere fibres (179 mmol kg(-1)) was 60% greater than in long-sarcomere fibres, due to higher concentrations of alanine, cysteine, glutamate, leucine and proline. The two fibre types exhibited differences in the banding pattern of the isoforms of troponin using gel electrophoresis. The average pool of amino acids in haemolymph was 2.7 mmol kg(-1). Cherax has symmetrical claws and the total pool of amino acids from whole muscles (approx. 79 mmol kg(-1)) was similar in left and right claw muscles. In animals acclimated to osmotic environments between 0 and 220 mOsm, the osmotic pressure of the haemolymph increased from 356 to 496 mOsm, but no systematic changes were observed in the amino acid profiles of muscles or haemolymph. The major findings were that (a) concentrations of amino acids differed between the two major fibre types in claw muscle and (b) amino acids in the muscle fibres did not play a major part in intracellular osmoregulation in Cherax, suggesting this species is an anisosmotic regulator.     

Taurine and β-alanine intraperitoneal injection in lactating mice modifies the growth and behavior of offspring

Taurine, one of the sulfur-containing amino acids, has several functions in vivo. It has been reported that taurine acts on γ-aminobutyric acid receptors as an agonist and to promote inhibitory neurotransmission. Milk, especially colostrum, contains taurine and it is known that milk taurine is essential for the normal development of offspring. β-Alanine is transported via a taurine transporter and a protein-assisted amino acid transporter, the same ones that transport taurine. The present study aimed to investigate whether the growth and behavior of offspring could be altered by modification of the taurine concentration in milk. Pregnant ICR mice were separated into 3 groups: 1) a control group, 2) a taurine group, and 3) a β-alanine group. During the lactation periods, dams were administered, respectively, with 0.9% saline (10?ml/kg, i.p.), taurine dissolved in 0.9% saline (43 mg/10?ml/kg, i.p.), or β-alanine dissolved in 0.9% saline (31 mg/10?ml/kg, i.p.). Interestingly, the taurine concentration in milk was significantly decreased by the administration of β-alanine, but not altered by the taurine treatment. The body weight of offspring was significantly lower in the β-alanine group. β-Alanine treatment caused a significant decline in taurine concentration in the brains of offspring, and it was negatively correlated with total distance traveled in the open field test at postnatal day 15. Thus, decreased taurine concentration in the brain induced hyperactivity in offspring. These results suggested that milk taurine may have important role of regulating the growth and behavior of offspring.     

Chemical changes in cell envelope and poly-β-hydroxybutyrate during short term starvation of a marine bacterial isolate

Qualitative and quantitative changes were observed in lipids, poly--hydroxybutyrate (PHB), and a cell wall peptidoglycan consitutent in a marine bacterial isolate during starvation for 24 h in an energy and nutrient-free medium. While the amount and composition of the membrane fatty acids fluctuated within the first hours of starvation, the total amount of fatty acids decreased during the starvation period. Furthermore, the ratio of monounsaturated to saturated fatty acids decreased and the proportion of short chain fatty acids increased. In the very early phase of starvation the bacteria contained PHB, which had been accumulated during the growth phase, but after 3 h no PHB was detected. Cells starved for phosphorus showed a different pattern as PHB was initially accumulated and did not decrease until 5 h of starvation. Synthesis of the cell wall amino acid d-alanine was initiated during the first phase of starvation. The effects of these changes on membrane fluidity and uptake of substrates as well as the use of fatty acids and PHB as energy resources during starvation are discussed.Non-common abbreviations FID flame ionization detector - GC gas chromatography - HFBA heptafluorobutyric anhydride - MS mass spectrometry - NSS nine salt solution - PHB poly--hydroxybutyrate - PFB pentafluorobenzylbromide     

Molecular characterization of D-amino acid oxidase from common carp Cyprinus carpio and its induction with exogenous free D-alanine

A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.     

Hepatopancreas gluconeogenesis during hyposmotic stress in crabs Chasmagnathus granulata maintained on high-protein or carbohydrate-rich diets

The incorporation of [14C]-alanine or [14C]-lactate into glucose was measured in hepatopancreas fractions from Chasmagnathus granulata crabs adapted to a high protein or a carbohydrate-rich diet and submitted or not (control group) to hyposmotic stress. Gluconeogenic capacity and phosphoenolpyruvate carboxykinase (PEPCK) activity increased during acclimation to a dilute medium in C. granulata hepatopancreas. In intact animals, high hemolymph urea levels occurred for the high-protein regimen and for crabs fed both diets and submitted to hyposmotic stress. It could be that the amino acids released during hyposmotic stress are deaminated in the hepatopancreas, and that the carbon chains are used as substrate for gluconeogenesis. Hepatopancreas gluconeogenesis seems to be one of the pathways implicated in the metabolic adjustment of the amino acid pool during hyposmotic stress in C. granulata.