Takehiko Yokoyama

Presence of Free D-Amino Acids in Microalgae

Several species of microalgae (phytoplankton), 4 species of freshwater algae and 4 species of marine diatoms, were cultured germ-free in the laboratory. The presence of free D-amino acids was verified in these species by a reversed-phase HPLC analysis. D-Aspartate was detected in all the microalgae examined, but D-alanine was only present in the marine diatoms. The D-amino acid content in Asterionella sp. of the marine diatoms increased from the exponential phase to the stationary phase and then decreased to the phase of decline.

Complementary DNA Cloning and Molecular Evolution of Opine Dehydrogenases in Some Marine Invertebrates

The complete complementary DNA sequences of genes presumably coding for opine dehydrogenases from Arabella iricolor (sandworm), Haliotis discus hannai (abalone), and Patinopecten yessoensis (scallop) were determined, and partial cDNA sequences were derived for Meretrix lusoria (Japanese hard clam) and Spisula sachalinensis (Sakhalin surf clam). The primers ODH-9F and ODH-11R proved useful for amplifying the sequences for opine dehydrogenases from the 4 mollusk species investigated in this study. The sequence of the sandworm was obtained using primers constructed from the amino acid sequence of tauropine dehydrogenase, the main opine dehydrogenase in A. iricolor. The complete cDNA sequence of A. iricolor, H. discus hannai, and P. yessoensis encode 397, 400, and 405 amino acids, respectively. All sequences were aligned and compared with published databank sequences of Loligo opalescens, Loligo vulgaris (squid), Sepia officinalis (cuttlefish), and Pecten maximus (scallop). As expected, a high level of homology was observed for the cDNA from closely related species, such as for cephalopods or scallops, whereas cDNA from the other species showed lower-level homologies. A similar trend was observed when the deduced amino acid sequences were compared. Furthermore, alignment of these sequences revealed some structural motifs that are possibly related to the binding sites of the substrates. The phylogenetic trees derived from the nucleotide and amino acid sequences were consistent with the classification of species resulting from classical taxonomic analyses.

A simple and rapid method for the analysis of fish histamine by paper electrophoresis

The described analytical method for histamine determination in fish and seafood consists of sample extraction, adsorption onto a paper disc, application of the paper disc onto electrophoresis paper, electrophoresis for only 10 min, drying, and color developing by Pauly’s reagent. Histamine can be satisfactorily detected and completely separated from histidine, carnosine and other Pauly reagent-positive compounds. This method does not require expensive instrumentation and any tedious pretreatment to eliminate potential interference by other imidazole compounds, such as histidine or carnosine. This method can be used to detect histamine in multiple fish and seafood samples simultaneously that contain as little as 15 p.p.m. histamine (1.5 mg/100 g).

Occurrence of β-alanine-specific opine dehydrogenase in the muscle of the limpet Cellana grata Gould (Archaeogastropoda)

The muscular tissues of the limpet Cellana grata exhibited beta-alanopine dehydrogenase (beta-AlDH) activity in addition to tauropine dehydrogenase (TaDH) activity and weak lactate dehydrogenase activity. Opine dehydrogenases (OpDHs) were purified, and two different types of OpDH, i.e. TaDHs and OpDHs showing beta-AlDH activity, were isolated. From the specificity for amino acid and opine, OpDHs showing beta-AlDH activity were concluded to be a true beta-AlDH showing strict substrate specificity for beta-alanine. Although the catalytic properties of beta-AlDH and TaDH were essentially similar, they were distinct from each other with respect to the amino acid substrate specificity and the K(m) values. Apparent K(m) values (mM) for the preferred amino acid substrate, pyruvate, NADH, the preferred opine substrate, and NAD+ were: 14.3 (beta-alanine), 0.19, 0.032, 35.2 (beta-alanopine), and 0.78 for beta-AlDH; and 33.3 (taurine), 0.53, 0.076, 48.6 (tauropine), and 0.58 for TaDH, respectively. Great similarities were found between beta-AlDH and TaDH with respect to molecular properties: molecular masses (both enzymes were monomeric proteins of approximately 40,000 Da), amino acid compositions, and N-terminal amino acid sequences (30 amino acid residues were identical). Partial similarities were also recognized between their lysyl endopeptidase maps. These results clearly show that beta-alanine-specific OpDH, a true beta-AlDH, is present in the limpet muscle.

Alanine racemase activity in the microalga thalassiosira sp.

In this paper, the authors report the detection of alanine racemase activity in the marine diatom Thalassiosira sp. Since the Thalassiosira sp. was cultured under germ-free conditions, it appeared that D-alanine was not derived from bacteria but was produced through catalysis by algal alanine racemase. The rate of conversion of L-alanine to D-alanine was approximately the same as that for the reverse reaction, and the enzyme catalyzed the equilibration of the D- and L-forms. The crude enzyme preparation obtained from the cells at the stationary phase of the growth cycle had an optimal pH of approximately 9.5. The Lineweaver—Burk analysis showed that the Km for D- and L-alanine was 16.5 mM and 29.4 mM, respectively. It appears that the enzyme is highly specific for D- or L-alanine because it does not catalyze the racemization of other amino acids. In addition, after gel filtration, the enzyme did not require exogenous pyridoxal 5′-phosphate (PLP) for its activity, however, the effects of several chemicals suggest that the enzyme may be PLP-dependent. The enzyme is more similar to that found in invertebrates when compared with that found in bacteria. This is the first report on the occurrence of alanine racemase activity in the microalga Thalassiosira sp.

Tauropine dehydrogenase from the starfish Asterina pectinifera (Echinodermata: Asteroidea): presence of opine production pathway in a deuterostome invertebrate.

Tauropine dehydrogenase (tauropine:NAD oxidoreductase; TaDH) was purified to homogeneity from the body wall of the starfish Asterina pectinifera Müller at Troschel(Echinodermata: Asteroidea) by means of (NH4)2SO4 precipitation followed by column chromatographies in DEAE-cellulose, Sephadex G75, Macro-prep ceramic hydroxyapatite, PBE 94, and Toyopearl HW50S. The enzyme was a monomeric protein of approximately 42000 Da and pI 5.2. The maximum rate of the tauropine biosynthetic reaction was observed at pH 6.0, and that of the tauropine catabolic reaction was at pH 8.7-9.2. Taurine and pyruvate were the preferred substrates. The tauropine catabolic reaction was inhibited by the substrate tauropine: the peak rate was observed at 12.5 mM. Apparent Km values for NADH, taurine, and pyruvate were 0.036 +/- 0.002, 21.3 +/- 1.6, and 0.46 +/- 0.02 mM, respectively, and for tauropine and NAD+ were 2.64 +/- 0.73 and 0.068 +/- 0.005 mM, respectively. The molecular and catalytic properties of the starfish TaDH were basically similar to those of TaDH from other species belonging to the lower invertebrate phyla and the middle phyla of Prostostomia. Tauropine accumulation in vivo during experimental anoxia was also demonstrated. These results gave clear evidence of opine production pathway in deutrostome invertebrate.

Biochemical and immunohistochemical analyses of GnRH-like peptides in the nerve ganglion of the chiton, Acanthopleura japonica.

We examined whether a gonadotropin-releasing hormone (GnRH)-like peptide is present in the nerve ganglion of the chiton Acanthopleura japonica (Mollusca, Polyplacophora) using reverse-phase high performance liquid chromatography (rpHPLC) combined with time-resolved fluoroimmunoas-say (TR-FIA) analysis, and immunohistochemistry. An extract of the chiton head region showed a similar retention time to that of synthetic lamprey GnRH-II on rpHPLC combined with TR-FIA analysis using a rabbit polyclonal antibody raised against chicken GnRH-II (aCII6). Cell bodies immunostained with LRH13 (a mouse monoclonal antibody raised against the common amino acid sequence of vertebrate GnRH) were detected in the cerebrobuccal ring (CBR). Cell bodies immunostained with aCII6 were not only observed in the CBR but also in the lateral nerve cord (LCo). Fibers immunostained with LRH13 and aCII6 were widely distributed throughout the central nervous system in the CBR, subradular ganglion (SubRG), pedal nerve cord (PCo), pedal commissure (P/PCom), lateropedal commissure (L/PCom), and from the LCo to the suprarectal commissure (SupRecCom). The cell bodies and fibers immunostained with these two antisera were distinguishable by dual-label immunohistochemistry. These results suggest that multiple GnRH-like peptides are present in the nerve ganglion of the chiton Acanthopleura japonica.

Immunohistochemical localization of endogenous D-Aspartate in the marine brown Alga Sargassum fusiforme.

Immunohistochemical localization (cellular localization) of endogenous D-aspartate in the marine brown alga Sargassum fusiforme was investigated by the use of a specific polyclonal antibody raised against D-aspartate. D-Aspartate immunoreactivity was evident in the medullary layer in the blade of the alga, and weak staining was found in the cortical layer, whereas epidermal cells were found to lack D-aspartate. Within the cells of the layers, immunoreactivity was confirmed only in the cytosol and not in the cell wall, chloroplast, or vacuole. These results suggest that D-aspartate is present in S. fusiforme cells, and excludes the possibility that it is derived from attached or symbiotic organisms such as marine bacteria. This is the first report describing the localization of free D-aspartate in plant cells.

Purification and some properties of alanine racemase from the marine gastropod Cellana grata

High levels of free D-alanine were found in the muscle of a marine gastropod Cellana grata that inhabited the intertidal zone, and alanine racemase activity was detected in the muscle. The authors purified alanine racemase from the muscle of C. grata to characterize its enzymological properties. The molecular mass of the enzyme was estimated to be 40.5 kDa by sodium dodecylsulfatepolyacrylamide gel electrophoresis and 41.4 kDa by gel filtration, suggesting that the enzyme was monomeric in nature. Kinetic experiments, performed using the purified enzyme, revealed that the Lineweaver-Burk plot for D-alanine as a substrate resulted in a Km value of 20.4 mM, and the value for L-alanine was 43.0 mM. Of the several types of amino acids tested, alanine was found to be the specific substrate for the enzyme. In the measurement of alanine racemase activity, exogenous pyridoxal 5′-phosphate (PLP) was not required for the enzyme activity; however, aminooxyacetic acid, hydroxylamine and phenylhydrazine, which inhibit PLP-dependent enzymes, strongly inhibited the enzyme activity. These results suggest that the enzyme is PLP-dependent. This is the first report on the purification and some properties of alanine racemase in a marine gastropod.

Changes in free amino acid concentrations and associated gene expression profiles in the abdominal muscle of kuruma shrimp Marsupenaeus japonicus acclimated at different salinities

ABSTRACT Shrimps inhabiting coastal waters can survive in a wide range of salinity. However, the molecular mechanisms involved in their acclimation to different environmental salinities have remained largely unknown. In the present study, we acclimated kuruma shrimp (Marsupenaeus japonicus) at 1.7%, 3.4% and 4.0% salinities. After acclimating for 6, 12, 24 and 72 h, we determined free amino acid concentrations in their abdominal muscle, and performed RNA sequencing analysis on this muscle. The concentrations of free amino acids were clearly altered depending on salinity after 24 h of acclimation. Glutamine and alanine concentrations were markedly increased following the increase of salinity. In association with such changes, many genes related to amino acid metabolism changed their expression levels. In particular, the increase of the expression level of the gene encoding glutamate-ammonia ligase, which functions in glutamine metabolism, appeared to be associated with the increased glutamine concentration at high salinity. Furthermore, the increased alanine concentration at high salinity was likely associated with the decrease in the expression levels of the the gene encoding alanine-glyoxylate transaminase. Thus, there is a possibility that changes in the concentration of free amino acids for osmoregulation in kuruma shrimp are regulated by changes in the expression levels of genes related to amino acid metabolism. Summary: Kuruma shrimp, Marsupenaeus japonicus, change free amino acid concentrations and associated gene expression levels in their muscle to adjust effectively to different salinities.