Tauropine dehydrogenase from the sandworm Arabella 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).

Occurrence of beta-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 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.

Nonradioactive assay for adenosine 5'-phosphosulfate sulfotransferase using reversed-phase ion-pair high-performance liquid chromatography.

A nonradioactive and simple method for the assay of adenosine 5'-phosphosulfate sulfotransferase (APSSTase) is described. The method is based upon the quantitative measurement of the substrate adenosine 5'-phosphosulfate and/or product AMP by ion-pair reversed-phase high-performance liquid chromatography on a C18 column. Isocratic elution renders the method simple and fast. This method is standardized for partially purified APSSTase from the marine red macroalga Porphyra yezoensis.

Occurrence of free D-aspartic acid in marine macroalgae.

The biologically rare D-aspartic acid was found in extracts of marine macroalgae. DL-aspartic acid was isolated from the Phaeophyta, Hizikia fusiformis, by ion-exchange chromatography, and crystallized from aqueous ethanol. It was characterized by optical-rotatory-dispersion analysis and reversed-phase HPLC analysis. The crystal consisted of equal amounts of D and L isomers. The presence of free D-aspartic acid was verified in 15 species from among 20 species of marine macroalgae by the same method (HPLC). Generally, Phaeophyta contained a high concentration of D-aspartic acid in contrast with the low concentration in Phycophyta and Rhodophyta. Particularly in Costaria costata, Hizikia fusiformis and Sargassum yezoensis, belonging to Phaeophyta, D-aspartic acid was found in concentrations proportional to those of L-aspartic acid.

Determination of octopine by pre-column fluorescence derivatization using benzoin.

A sensitive fluorimetric method for the determination of octopine, a member of opine family, is presented. The method is based on the formation of a fluorescent derivative of octopine with benzoin and the separation by high performance liquid chromatography using a reversed-phase column (Kaseisorb LC ODS-300) within 20 min. The octopine derivative is completely separated from other guanidino compounds including arginine which is generally very high in marine invertebrates. This method gives higher sensitivity, 5 pmol minimum detection, and better reproducibility than the electrophoresis method and colorimetric method.

Specific determination of histamine in fish by high-performance liquid chromatography after diazo coupling.

A new approach to the pre-column derivatization and analysis of histamine by HPLC is described, the method being based upon the formation of a diazo-coupled derivative of histamine. This derivatization method is simple, efficient, sensitive and specific for histamine and other imidazole compounds without a preliminary clean-up step. The liquid chromatographic system allows for rapid, bonded-phase separation with visible-light detection of these compounds within 20 min at a 10-pmol sensitivity. By using this method, the production of a large amount of histamine was confirmed in the muscle of mackerel Scomber japonicus incubated with the intestinal contents of the mackerel.

Effects of octopine on the serum cholesterol level in rats.

The effects of dietary octopine, which is one of the major extractive component of marine molluscs, on the level of serum and liver cholesterol of rats fed with cholesterol-enriched or cholesterol-free diets were investigated. Dietary supplementation with 1.5% octopine in a cholesterol-enriched diet significantly decreased the serum total- and VLDL+LDL-cholesterol levels and by contrary increased the serum HDL-cholesterol level in rats. The same tendency was observed in the rats fed with 1.5% octopine in a cholesterol-free diet.

Electroporation as a new technique for producing transgenic fish.

A recombinant plasmid, pMV-GH, containing rainbow trout growth hormone cDNA fused to mouse metallothionein I promoter, was introduced into medaka (Oryzias latipes) by electroporation. Of 3109 fertilized eggs treated with electric pulses (750 V/cm, 50 microseconds, 5 times), 783 (25%) hatched out. Four percent of the hatchlings were transgenic. To obtain transgenic lines, 180 hatchlings were maintained and 35 of them grew into adult fish. Two of these fish were transgenic. When one transgenic fish was mated with a normal female, the transgene was found in all the F1 offspring assayed. In F2 offspring obtained by mating transgenic F1 fish, 88% were transgenic.