Nobuhiro Kanno

Distribution of opine dehydrogenases and lactate dehydrogenase activities in marine animals

Abstract 1. 1. Opine dehydrogenases (OpDHs) and lactate dehydrogenase (LDH) activities were determined in various marine animals. OpDHs were detected in six marine invertebrate phyla; Porifera, Coelenterata, Annelida, Mollusca, Arthropoda and Echinodermata in phylogenic sequence. 2. 2. Among several OpDHs, tauropine dehydrogenase (TaDH) occurred widely in marine invertebrates, from Porifera to Echinodermata. 3. 3. With a few exceptions, total OpDHs activities exceeded that of LDH activity in the marine invertebrates investigated. 4. 4. With respect to anaerobic glycolysis, OpDHs are indicated to play an important role in phylogenically lower invertebrates, whereas LDH is more important in higher animals.

Distribution of neuroexcitatory amino acids in marine algae

Abstract Contents of three neuroexcitatory amino acids, N- methyl- d,l -aspartic acid (NMA), kainic acid (KA) and domoic acid (DA) were investigated in 46 marine algae species. NMA was detected in very small amounts in several marine algae belonging to the Chlorophyceae and Rhodophyceae, but not in algae belonging to the Phaeophyceae. KA was detected in three species and DA was detected in four species all belonging to the family Rhodomelaceae (Ceramiales, Rhodophyceae). Among rhodomelacean algae investigated in this study, KA and DA were detected only in the particular species collected in the south-west Islands of Japan (Tokunoshima Island and Okinawa Main Island). It is noteworthy that the distribution of both KA and DA was strictly restricted some algae of the subtropical area.

Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta): stabilization, purification, and properties

Adenosine 5′-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel-filtration. The native Mr measured by gel-filtration was 350 000. The subunit Mr was estimated to be 43 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In addition, APSSTase had a relatively broad pH optimum of pH 9.0–9.8 with a peak at pH 9.5. The apparentKm value for adenosine 5′-phosphosulfate (APS) was 2.1 μM, when dithiothreitol was acceptor substrate. 3′-Phosphoadenosine 5′-phosphosulfate and inosine 5′-phosphosulfate could not substitute for APS as a sulfate donor. The enzyme utilized several organic thiols as acceptor substrates (artificial substrates): dithiothreitol (apparentKm = 1.5 mM) and dithioerythritol (apparentKm = 1.5 mM) gave the highest activity, and appreciable activity was also obtained usingl-glutathione (reduced form) which exhibited slight substrate inhibition (apparentKm = 0.6 mM; the initial velocity was maximal at 3.0–4.0 mM). While APSSTase was markedly unstable in vitro: the half-life for activity loss at 25°C and pH 9.5 was about 8 min, the instability was decreased in the presence of a relatively high concentration of Na2SO4 or (NH4)2SO4, and in the presence of APS or its analogs (AMP and β-methylene-APS). Most of the thiols, with the sole exception of glutathione, were found to inactivate APSSTase irreversibly. The thiol-mediated inactivation was completely inhibited by the high concentration of Na2SO4, and by the analogs of APS.

Quantitative HPLC analysis of acidic opines by phenylthiocarbamyl derivatization

A new method for the quantitative analysis of acidic opines--alanopine, strombine, tauropine, and beta-alanopine--is presented. The method is based on formation of phenylthiocarbamyl (PTC) derivatives of the acidic opines after partial purification by ion-exchange chromatography. The PTC acidic opines are separated by reverse-phase high-performance liquid chromatography (HPLC) and detected within 20 min by ultraviolet absorbance. This HPLC method gives higher sensitivity, 10-30 pmol minimum detection, and better reproducibility than the high-voltage paper electrophoresis method. There is also good agreement for the three acidic opines (alanopine, strombine, and tauropine) when compared by HPLC and electrophoresis methods. Accumulation of beta-alanopine was observed for the first time in the adductor muscle of blood shell, Scapharca broughtonii, during aerial exposure by application of the HPLC detection method.

Isolation of a new opine, β-alanopine, from the extracts of the muscle of the marine bivalve mollusc, Scapharca broughtonii

Abstract 1. 1. A new opine compound, β-alanopine [ N-( d -1- carboxyethyl )-β- alanine ] was isolated from the muscle of the blood shell, Scapharca broughtonii. 2. 2. β-Alanopine dehydrogenase activity, which catalyzes the reductive condensation of β-alanine and pyruvate, was confirmed in this bivalve mollusc. 3. 3. Since tissues of this bivalve showed a high β-alanopine dehydrogenase activity and contained large amounts of β-alanine, β-alanopine dehydrogenase may play an important physiological role in the regulation of cytoplasmic redox balance like other opine dehydrogenases.

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.

Tauropine dehydrogenase from the sandwormArabella iricolor (Polychaeta: Errantia): Purification and characterization

This is the first report of the purification of tauropine dehydrogenase (NAD: tauropine oxidoreductase) from a polychaete worm. In the sandworm Arabella iricolor Montagu (Polychaeta: Errantia), two forms of TaDH were detected: major component (pl = 7.5) and minor one (pl = 6.4). The major TaDH component was purified to homogeneity by means of (NH4)2SO4 precipitation, anion-exchange, affinity, chromatofocusing and hydrophobic chromatography, and characterized. From the molecular mass of 43.7 kDa obtained by rapid gel-filtration and that of 43.5 kDa by SDS-PAGE, the sandworm enzyme appeared to be a monomeric protein. Maximum rates of reduction of pyruvate and oxidation of tauropine were observed at pH 7.0 and 8.5, respectively. Pyruvate and taurine were preferred substrate for the enzyme. Apparent K(m) values determined using constant co-substrate concentrations were: 35.7 mM, 0.34 mM, and 0.036 mM for taurine, pyruvate and NADH, respectively, in the tauropine synthesizing reaction; and 4.8 mM and 0.051 mM for tauropine and NAD+, respectively, in the tauropine oxidizing reaction. The tauropine synthesizing reaction was subject to substrate inhibition by pyruvate: maximum rate was observed at 2.5-3.0 mM (inhibitory range of pyruvate concentration producing half-maximal rate was 26.8 mM).