N-Methyl-d -glutamate and N-methyl-l -glutamate in Scapharca broughtonii (Mollusca) and other invertebrates

Comparative Biochemistry and Physiology Part B 134 (2003) 79–87

N-Methyl-D-glutamate and N-methyl-L-glutamate in Scapharca broughtonii (Mollusca) and other invertebrates Atsuko Taruia, Kimihiko Shibataa,c, Shouji Takahashia, Yoshio Keraa, Toratane Munegumib, Ryo-Hei Yamadaa,* a Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan Department of Materials Chemistry and Bioengineering, Oyama National College of Technology, Oyama, Tochigi 323-0806, Japan c Biological Information Research Center, National Institute of Advanced Industrial Science and Technology. Koto-ku, Tokyo 135-0064, Japan

b

Received 16 July 2002; received in revised form 1 September 2002; accepted 25 September 2002

Abstract The presence of N-methyl-D-glutamate (NMDG) and N-methyl-L-glutamate (NMLG) has been demonstrated in the tissues of Scapharca broughtonii, which are known to contain N-methyl-D-aspartate (NMDA). To our knowledge, this is the first report on the natural occurrence of NMDG and the occurrence of NMLG in eukaryotes. These compounds were identified according to the following findings; (a) their derivatives with (q)- and (y)-l-(9-fluorenyl)ethyl chloroformate (FLEC) showed identical behaviors with those of authentic NMDG and NMLA, respectively, on highperformance liquid chromatography (HPLC), (b) the HPLC peak of NMDG disappeared when the extract, as well as the authentic compound, was treated with D-aspartate oxidase before derivatization, (c) they behaved identically with authentic compounds on thin-layer chromatography and differently from NMDA. Both or either of NMDG and NMLG were also detected in several mollusks and other animals. Concentrations of the enantiomers were comparable in the tissues of S. broughtonii and a few other species. 䊚 2002 Elsevier Science Inc. All rights reserved. Keywords: N-Methyl-D-aspartate; N-Methyl-D-glutamate; N-Methyl-L-glutamate; Mollusks; Scapharca broughtonii

1. Introduction N-Methyl-D-aspartate (NMDA) has attracted extensive attention because of its specific action as an agonist for a type of glutamate receptor in the central nervous system of higher animals. This compound, first known as an artificial compound, was discovered to be a natural product since it was isolated from the foot muscle of blood shell Scapharca broughtonii (Sato et al., 1987). We *Corresponding author. Tel.: q81-25-847-9635; fax: q8125-847-9635. E-mail address: [email protected] (R.H. Yamada).

developed a high-performance liquid chromatographic (HPLC) method to determine this compound rapidly with small amounts of tissue samples (Todoroki et al., 1999) and demonstrated its occurrence in several bivalves (Shibata et al., 2001). During these studies, we also found some other compounds that were similar to NMDA in HPLC behavior. One of them found in Corbicula sandai and Tapes japonica was identified as Nmethyl-L-aspartate (Shibata et al., 2001). In the present paper, we report that two of them found in S. broughtonii in addition to NMDA are Nmethyl-D-glutamate (NMDG) and N-methyl-L-glutamate (NMLG). We also present the distribution

1096-4959/03/$ - see front matter 䊚 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 9 6 - 4 9 5 9 Ž 0 2 . 0 0 2 3 1 - 2

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A. Tarui et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 79–87

of these compounds in various mollusks and other animals. 2. Materials and methods 2.1. Animals Live specimens of the following animals were purchased at local fish markets in Niigata Prefecture, Japan: (a) mollusks Bivalvia: ark shell S. broughtonii, half-crenate ark shell Scapharca subcrenata, Asian hard clam Meretrix lusoria, blue mussel Mytilus edulis, Sakhalin surf clam Pseudocardium sachalinensis, ezo giant scallop Patinopecten yessoensis, oyster Crassostrea nippona and tellins Peronidia venulosa, (b) mollusks Gastropoda: horned turban shell Batillus cornutus, whelk Buccinum striatissimum, ezo whelk Buccinum middendorffi and ezo neptune whelk Neptunea polycostata, (c) mollusks Cephalopoda: octopus Paroctopus conispadiceus and pacific flying squid Todarodes pacificus, (d) prochordates Urochordata: sea-squirt Halocynthia roretzi. Live specimens of echinoderms Echinoidea: sea urchin Anthocidaris crassispina were collected from a beach in Niigata Prefecture, Japan. The tissues of the animals were removed and washed with ice-cold saline, and were stored at y30 8C until use. The foot and mantles of S. broughtonii were divided into outside and inside parts as described previously (Todoroki et al., 1999). 2.2. Materials (q)-l-(9-Fluorenyl)ethyl chloroformate w(q)FLECx and (y)-l-(9-fluorenyl)ethyl chloroformate w(y)-FLECx were purchased from Aldrich (Milwaukee, WI, USA). Solvents for HPLC, such as acetonitrile, tetrahydrofuran and methanol of HPLC grade were obtained from Nacalai Tesque (Kyoto, Japan). N-Methyl-DL-glutamate, NMLG and NMDA were purchased from Sigma (St. Louis, MO, USA). The anion-exchange resin, Dowex 1=2 (100–200 mesh, chloride form) was obtained from Muromachi Chemicals (Tokyo, Japan) and converted to acetate form just before use. A reverse-phase HPLC column (250=4.6 mm i.d.), packed with 4 mm-diameter J9 sphere ODSM80 was from YMC (Kyoto, Japan). Other chemicals were of analytical purity.

2.3. Preparation of tissue extracts The frozen tissues (usually approx. 0.3 g) were thawed and homogenized with a high-speed microhomogenizer (Physcotron, Niti-on, Funabashi, Japan) or a Potter-Elvehjem homogenizer equipped with a Teflon pestle in 10 volumes of 8% perchloric acid under cooling in ice water. The homogenate was centrifuged for 20 min at 12 000=g, at 4 8C, and the supernatant was neutralized with KOH. After centrifugation to remove the precipitated KClO4, the supernatant (2–5 ml) was loaded on a column (2 ml volume) of Dowex 1=2 (100– 200 mesh, acetate form), and the column was washed with 20 ml of water to remove neutral and basic substances. N-Methylglutamates and acidic amino acids including glutamate enantiomers were eluted with 20 ml of 1 M acetic acid. The eluate was concentrated to dryness under reduced pressure with a centrifugal evaporator (VC-960, Taitec, Saitama, Japan) at 40 8C. The residue was subjected to analysis by HPLC. 2.4. HPLC analysis 2.4.1. Determination of N-methyl-D,L-glutamate and N-methyl-D,L-aspartate The derivatization of acidic N-methyl amino acids and the chromatography were performed according to our previous report (Todoroki et al., 1999) as follows: The residue, resulting from the procedure of Section 2.3 described above, was dissolved in 2–3 ml of 0.1 M sodium borate buffer (pH 9.0). A 20 ml volume of this sample solution was mixed, in a 1.5 ml microcentrifuge tube, with 10 ml of o-phthalaldehyde (OPA) solution (5 mgy ml) in acetonitrile and was kept at 50 8C for 15 min to remove primary amino acids. Then, 5 ml of 18 mM (q)- or (y)-FLEC in acetone and 5 ml of acetonitrile were added and, after reaction for 15 min at 50 8C, a 10 ml volume of 100 mM aqueous L-cysteic acid in 0.1 M sodium borate buffer (pH 9.0) was added to remove the remaining FLEC. The mixture was allowed to react for another 7 min at the same temperature. After addition of 150 ml of 0.1 M sodium acetate buffer (pH 4.0), the mixture was filtered through a 0.45mm filter (Nacalai Tesque) and injected into the HPLC system. The injection volume was 20–100 ml.

A. Tarui et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 79–87

The HPLC analysis was performed with a Shimadzu (Kyoto, Japan) HPLC system consisting of two LC-10AD pumps, a SIL-10A auto injector equipped with a sample cooler S (3 8C), a CTO10A column oven, a SCL-10 A system controller, a DUG-3A degasser, an RF-10A fluorescence detector and a Chromatopak C-R5A data processor. The analytical column was a reversed-phase Jsphere ODS-M80 (250=4.6 mm i.d.)(YMC, Kyoto, Japan). Unless otherwise stated, the mobile phase consisted of 86% (vyv) 0.1 M sodium acetate buffer (pH 5.59), 7% (vyv) acetonitrile and 7% (vyv) tetrahydrofuran, and isocratic elution was carried out at a flow-rate of 1.2 mlymin and at a column temperature of 40 8C. For fluorometric detection of eluted FLEC derivatives the excitation and emission wavelengths were set at 260 and 315 nm, respectively. While authentic NMLG and NMDA were commercially available and served as HPLC standards, NMDG was not. We, therefore, used the NMDG in the commercially available racemic N-methylD,L-glutamate as an HPLC standard, on assumption that the racemate is an exactly equimolar mixture of enantiomers. Comparison of the authentic NMLG and the racemate in the HPLC behavior enabled us to identifiy the peak of NMDG. 2.4.2. Determination of D,L-glutamate The residue, resulting from the procedure of Section 2.3 described above, was dissolved in 2– 5 ml of 100 mM L-cysteic acid solution (adjusted to approximately pH 7.0 with KOH), which was employed as the internal standard. The derivatization of amino acids with OPA and N-acetyl-Lcysteine was performed by the method of Aswad (Aswad, 1984). The HPLC analysis was carried out according to our previous study (Watanabe et al., 1998) with several modifications, using a Shimadzu (Kyoto, Japan) HPLC system consisting of two LC-6A pumps, an RF-10A fluorescence detector, a Chromatopac C-R4A data processor, a DGU-3A degasser and a J’ sphere-ODS-M80 column (250=4.6 mm i.d.) (YMC, Kyoto, Japan). Excitation and emission wavelengths were 320 and 440 nm, respectively. Gradient elution was performed with 3.5–5.5% (0–45 min) methanol in 50 mM sodium acetate buffer (pH 5.53) at a flow rate of 1.0 mlymin. Peaks were identified by

81

comparison with the behaviors of authentic D- and L-glutamate.

2.5. Thin-layer chromatography of tissue extracts The identity of N-methylglutamate enantiomers in the tissue extracts was further confirmed by thin-layer chromatography (TLC) as follows: The residue, resulting from the procedure of Section 2.3 described above, was dissolved in 0.1 M sodium borate buffer (pH 9.0). A 200 ml portion of the solution was applied onto a precoated Merck 25 TLC plastic sheet (Silica gel 60, 20=20 cm) (Merck, Darmstadt, Germany) by streaking in a width of 5 cm. The sheet was developed in a solvent system: n-butyl alcohol–acetic acid–water (3:1:1, vyv) at room temperature. After the first run, the sheet was dried by standing and developed again with the same solvent. This procedure was repeated until the fifth development was finished, and the migration positions of N-methylglutamates and N-methylaspartates were identified by the behavior of authentic N-methyl-D,L-glutamate and N-methyl-D,L-aspartate run in parallel on the same plate and visualized by the ninhydrin method for detection of N-methyl amino acids (Dawson et al., 1986). The portion of the plate where the silica gel contained N-methylglutamates was cut out, and the compounds were extracted from the gel with 63% ethanol. The extract was concentrated to dryness under reduced pressure with a centrifugal evaporator at 40 8C. The residue was redissolved in 100 ml of 0.1 M sodium borate buffer (pH 9.0). The solutions were injected into the HPLC after derivatization with (q)-FLEC or (y )-FLEC as described above (Section 2.4.1.). 2.6. Treatment of tissue extracts and authentic compounds with D-aspartate oxidase A mixture of 400 ml of a tissue extract or authentic compounds in 0.1 M sodium borate buffer (pH 9.0), 20 ml of 0.5 mM FAD, 10 ml of catalase (2.5 mgyml), 40 ml of 300 mM sodium pyrophosophate buffer (pH 8.0) and 30 ml of Daspartate oxidase (7.68 mmolymin per ml) from pig kidney (Yamada et al., 1996) was incubated at 37 8C for 4 h. The reaction was stopped by addition of 4.5 ml of 16% perchloric acid. The supernatant was neutralized with KOH and, after removal of precipitates, concentrated to dryness.

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Fig. 1. HPLC chromatograms of authentic NMDG and NMLG derivatized with (q)-FLEC (a) and (y)-FLEC (b), and a tissue extract of S. broughtonii derivatized with (q)-FLEC (c) and (y)-FLEC (d). For (a) and (b), a mixture of 10 pmol NMDG and 15 pmol of NMLG derivatized with (q)- or (y)-FLEC was injected into the HPLC system. For (c) and (d), the extract was prepared from the outside part of foot, and a portion of the extract corresponding to 0.5 mg tissue was derivatized with (q)- or (y)-FLEC and injected. Peaks: 1; L-cysteic acid, 2 and 29; (q)- and (y)-FLEC derivatives of NMDG, 3 and 39; (q)- and (y)-FLEC derivatives of NMLG, 4; NMDA.

The residue was dissolved in 0.1 M sodium borate buffer (pH 9.0) to be subjected to the precolumn derivatization process. 3. Results 3.1. Occurrence of N-methylglutamate enantiomers in the foot muscle of S. broughtonii Fig. 1a shows a chromatogram of (q)-FLEC derivatives of authentic NMDG and NMLG, which were clearly resolved under the HPLC condition employed. When (y)-FLEC was used in place of (q)-FLEC as derivatizing agent, the retention times of NMDG and NMLG were exactly reversed as expected (Fig. 1b). Fig. 1c shows a typical chromatogram of the extract of S. broughtonii foot muscle derivatized with (q)-FLEC. Two peaks (peak 2 and 3) following that of NMDA exhibited,

respectively, the same retention times as those of authentic NMDG and NMLG in Fig. 1a. Addition of authentic compounds to the extract before derivatization only made these peaks higher and did not produce any other new peaks. When the extract was derivatized with (y)-FLEC, the chromatogram (Fig. 1d) showed, in addition to a large peak at the right end, two peaks, one of which (peak 29) apparently represented NMDG and the other were likely to represent NMLG combined with NMDA. To obtain further evidence, we treated the extract as well as authentic N-methylglutamate enantiomers with pig kidney D-aspartate oxidase, which is known to oxidize NMDA as one of its specific substrates. The enzyme treatment completely degraded authentic NMDG and not NMLG, confirming that NMDG is also a substrate of this oxidase (Fig. 2a,b) as shown by preliminary exper-

A. Tarui et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 79–87

83

Fig. 2. Effect of D-aspartate oxidase treatment on authentic NMDG and NMLG, and a tissue extract of S. broughtonii. Authentic compounds treated (b) and untreated (a) with D-aspartate oxidase, and an extract of the outside part of foot treated (d) and untreated (c) with the enzyme were derivatized with (q)-FLEC and subjected to HPLC. For (a), 25 pmol NMDG and 25 pmol of NMLG derivatized were in an injected sample into the HPLC system. A mixture of 50 nmol NMDG and 50 nmol NMLG for (b), or an extract from 40 mg tissue for (d) was treated with D-aspartate oxidase and processed as described in Section 2.6. One percent of the resulting extract was used for a derivatized sample injected. For (c), the same extract as for (d) was processed in the same way except that the enzyme treatment was stopped with perchloric acid without incubation. Peaks: 1; L-cysteic acid, 2; NMDG, 3; NMLG, 4; NMDA.

iments (data not shown). Similarly, Fig. 2c,d show that the enzyme treatment abolished the peak 2 as well as the peak of NMDA (peak 4) and not the peak 3 in the chromatogram of the extract. All these data support that the peaks 2 and 3 obtained from the extract represent the FLEC derivatives of NMDG and NMLG. To confirm the identity of these N-methylglutamates, we examined their behavior on TLC. We subjected the extract to TLC and cut out a portion of the plate that was expected to contain Nmethylglutamates as indicated by the position of the authentic racemic compounds (12.5 cm from the origin), which migrated faster than racemic Nmethylaspartates (10.8 cm from the origin). Then, the extract from the gel on the cut-out plate was examined by HPLC. The chromatogram (Fig. 3b) showed two peaks of NMDG and NMLG whose area accounted for approximately 80% of those given by the extract that did not undergo TLC

(Fig. 3a). In addition, the peak of NMDA observed in Fig. 3a was not shown in Fig. 3b. 3.2. Distribution of N-methylglutamate enantiomers in the tissue of various mollusks and other animals compared with that of D,L-glutamate Table 1 shows the distribution of NMDG and NMLG as well as that of D- and L-glutamate. Many tissues of S. broughtonii other than foot also contained NMDG and NMLG at comparable concentrations with those in the foot. The D y(DqL) ratio was likewise approximately 50%. Both of the N-methylglutamate enantiomers were detected in some of the other organisms: bivalves such as Scapharca subcrenata, Meretrix lusoria, Mytilus edulis and Spisula sachalinensis; snails such as Batillus cornutus, Buccinum middendorffi and; octopus Paroctopus conispadiceus; sea urchin Anthocidaris crassispina; and sea-squirt Halocyn-

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All of the tissues examined contained both of and L-glutamate. The D y(DqL) ratio was generally below 1% and at highest a few percent, apparently independent of the ratio for N-methylglutamate. In the same tissues, the D y(DqL) ratio of aspartate determined in parallel (data not shown) was much higher in agreement with previous reports(Shibata et al., 2001; D’Aniello and Giuditta, 1977, 1978). D-

4. Discussion

Fig. 3. HPLC of N-methylglutamates in a tissue extract of S. broughtonii purified by thin layer chromatography. An extract of the outside part of the foot was examined by HPLC with (q)-FLEC as a derivatizing agent, before (a) and after (b) thin layer chromatography. For (a), a portion of the extract corresponding to 1 mg tissue was derivatized and injected. For (b), an extract from 20 mg tissue was subjected to thin layer chromatography and compounds associated with the layer at the same migration distance as that of authentic N-methyl-D,Lglutamate were extracted. Five percent of the resulting extract was injected after derivatization. Peaks: 1; L-cysteic acid, 2; NMDG, 3; NMLG, 4; NMDA.

thia roretzi. Among these species, Neptunea polycostata was similar to S. broughtonii in the level of highest NMDG content and in that most of the tissues contained the enantiomers with the D y(Dq L) ratio of approximately 50%. In the other species, their presence was often limited to certain tissues and their levels were generally lower with the D y(DqL) ratio widely scattered. Only one of the enantiomers was detected in some organisms: NMDG was found in Crassostrea nippona, while NMLG in Patinopecten yessoensis, Peronidia venulosa and Todarodes pacificus. Neither enantiomer was significantly detected in other animals examined including crustaceans, mammals and fishes (data not shown).

In this work we have presented the following findings that support the occurrence of NMDG and NMLA in S. broughtonii: (a) the tissue extract contained compounds which exhibited identical behaviors on HPLC with those of authentic NMDG and NMLG, when they were derivatized with (q )-FLEC and (y)-FLEC; (b) the HPLC peak of NMDG was abolished when the extract as well as authentic NMDG was treated with D-aspartate oxidase before derivatization; (c) the compounds in the extract behaved identically with authentic compounds on TLC and differently from NMDA. With these findings taken together, there seems to be little doubt about the identification, although studies with mass spectroscopy would provide more conclusive evidence. To our knowledge, this is the first report on the natural occurrence of NMDG and the occurrence of NMLG in eukaryotes, since NMLG was reported to be produced by methylation of L-glutamate in certain bacteria (Hersh, 1985). The finding that both enantiomers occur in several species contrasts with the previous report that there was no mollusk species containing both of NMDA and NMLA (Shibata et al., 2001). Moreover, both NMDG and NMLG have been found in N. polycostata where neither of the Nmethyl aspartates was detected. In addition, at least either of the N-methylglutamate enantiomers has been detected in many species containing neither of the N-methylaspartates. These findings appear to suggest a wider distribution of N-methylglutamates in animals and their more common roles. In view of the finding that S. broughtonii possesses aspartate racemase as well as comparable amounts of D- and L-aspartate (Watanabe et al., 1998), it seems possible that N-methylglutamates are also racemized in this organism. The very low D y(DqL) ratio observed for glutamate independent of the ratio for N-methylglu-

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85

Table 1 Distribution of N-methylglutamates and free glutamates in the tissues of mollusks and other invertebrates Animals

N-Methylglutamate n

D

form

Glutamate L

form

(nmolyg tissue) MOLLUSCA, Bivalvia Scapharca broughtonii Foot (outside) Foot (inside) Mantle (outside) Mantle (inside) Adductor Gill Kidney Labial palp Liver Venticle Anus Posterior pedal retractor Scapharca subcrenata Foot Mantle Adductor Gill Viscera Meretrix lusoria Foot Mantle Adductor Siphon Viscera Mytilus edulis Mantle Spisula sachalinensis Mantle Patinopecten yessoensis Foot Mantle Crassostrea nippona Foot Viscera Peronida venulosa Foot Mantle MOLLUSCA, Gastropoda Batillus cornutus Foot Mantle Gill Muscle Liver Buccinum middendorffi Foot Mantle Gill Muscle Tentacle Snout Viscera Neptunea polycostata Foot Mantle

D y(DqL)

n

D

form

L

form

(nmolyg tissue)

%

D y(DqL)

%

14 3 3 3 3 3 2 2 3 1 1 3

17.9"14.1 25.7"17.1 23.5"22.3 10.9"6.29 15.2"3.57 15.9"10.1 10.3, 10.2 2.30, 3.70 5.00"2.80 4.60 7.50 7.70"2.20

13.9"12.2 5.29"1.24 7.80"5.74 12.8"8.18 6.09"3.38 9.27"2.17 4.70, 4.50 2.20, 12.7 2.80"2.00 2.7 5.1 4.10"2.40

55.1"26.0 80.2"9.2 67.7"16.3 48.7"27.6 72.0"11.3 57.4"26.3 68.6, 69.4 50.4, 22.5 68.1"9.8 62.9 59.7 66.4"10.2

14 3 3 3 3 3 2 2 3 1 1 3

29.5"21.3 56.3"26.8 27.3"15.7 25.4"8.51 57.0"23.8 33.5"18.6 358, 221 nd 164"52.1 165 nd 122"62.0

5690"2710 4970"68.3 5760"1380 4530"1070 9450"484 5610"729 15800, 13700 nd 13100"2830 13500 10300 16000"2240

0.498"0.199 0.600"0.267 0.472"0.255 0.566"0.165 0.600"0.247 0.69"0.273 2.20, 1.60

3 3 3 3 2

0.87"0.67 1.78"1.28 1.45"0.23 q q

2.23"0.70 6.20"0.60 2.53"0.16 9.59"10.6 nd, 2.95

25.8"20.7 20.4"13.3 34.8"5.5

3 3 3 3 2

47.8"18.4 63.2"50.4 45.3"32.6 117"73.6 31.3, 171

3790"391 3115"435 2860"688 2710"607 4070,3910

1.24"0.48 2.11"1.90 1.41"0.90 4.13"1.89 0.764, 4.20

3 3 3 3 2

nd nd nd nd q, 5.41

q 4.05"0.61 1.16"1.17 q 26.5, 24.3

18.2

3 2 3 3 3

80.1"49.2 10.1, 14.6 19.9"19.1 20.3"8.04 40.2"12.0

15700"13200 3820, 4160 5000"2420 4350"1240 9100"1660

0.570"0.134 0.307, 0.062 0.349"0.175 0.523"0.333 0.443"0.114

2

2.29, nd

2.86, 2.44

30.2

2

124, 155

4010, 4590

3.01, 3.27

2

0.82, 0.84

1.31, nd

38.6

2

50.1, 46.5

4390, 4360

1.13, 1.05

2 3

nd nd

nd 2.05, 0.71

3 3

28.0"10.4 20.0"7.25

2300"326 2310"458

1.26"0.58 0.842"0.167

3 3

nd 2.70"0.51

nd nd

3 3

35.2"21.5 18.4"12.6

4970"68.3 3040"2140

0.707"0.435 0.669"0.525

3 3

nd nd

3 3

13.4"1.07 3.87"4.62

1130"1350 614"306

2.64"1.88 0.715"0.841

3 3 3 3 3

nd 0.79"0.36 nd nd nd

nd 6.82"5.45 nd nd nd

3 3 3 3 3

50.6"70.4 10.1q11.7 76.5"71.3 19.0"18.9 40.8"27.2

3320"2880 1050"714 4840"3000 2550"1750 4210"1700

1.04"0.94 0.841"0.514 1.16"1.11 0.598"0.679 0.861"0.396

2 2 2 3 1 3 2

nd nd 0.38, 0.36

nd nd 24.0,11.8 nd 3.11 nd 4.89, 8.47

3 4.89, 8.47 nd 31.4 nd 24.0,11.8

3 2 2 3 1 3 3

8.15, 22.2 27.8, 15.6 18.2, 25.9 20.5"11.2 16.3 133"212 29.7"14.4

1950, 2700 2020, 3940 5250, 5120 3440"2060 2530 1230"1420 4580"50.0

0.416"0.814 1.35, 0.39 0.345, 0.529"0.136 0.641 1.06"0.90 0.645"0.312

35.7"13.0 37.8"11.0

3 3

34.1"12.8 29.7"2.83

6840"1870 6520"614

0.537"0.073 0.378"0.048

3 3

1.43 155,1.13 1.89"1.35 2.78"1.14

1.35"0.68 1.26"1.12

2.39"1.43 4.42"1.13

17.4"15.2

1.22"0.13 1.20 0.724"0.287

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A. Tarui et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 79–87

Table 1 (Continued) Distribution of N-methylglutamates and free glutamates in the tissues of mollusks and other invertebrates Animals

N-Methylglutamate n

D

form

Glutamate L

form

(nmolyg tissue) Gill Tentacle Ovary Kidney Liver Viscera Buccinum striatissimum Snout

1.39"0.61 4.22"3.32 0.434"0.220 27.8"11.4 2.49"1.36 3.23"2.82

2

2.13, 2.23

2 2 1 2 2 2 2 2

n

68.2"32.5 45.3"13.4 42.1"7.20 94.1"1.6 61.0"16.6 50.8"20.3

form

L

form

(nmolyg tissue)

% 1.29"0.69 4.19"1.38 0.56"0.215 1.64"0.42 1.36"0.34 2.39"0.61

D

D y(DqL)

%

3 3 3 3 3 3

50.3"35.5 42.4"3.14 30.8"4.60 59.1"15.7 25.3"5.39 27.5"3.36

q

2

1.75, 2.50

nd

nd nd nd nd nd nd nd nd

nd nd 8.15 q nd nd nd nd

2 2 1 2 2 2 2 2

327, 215 197, 42.1 48.9 49.5, 21.2 23.9, 16.6 18.1,5.22 21.7, 3.59 19.6, 5.50

11600, 4920 13000, 5300 4030 2150,5650 907,819 899, 823 627, 1030 2830, 222

3 3 3 3 3

nd q nd q q

nd nd nd q 0.384"0.064

3 3 3 3 3

137"78.0 66.0"22.7 38.9"1 9.8 42.6"20.7 20.8"1 0.1

5260"795 5230"160 3020"994 3250"1640 3840"683

2.49"1.22 1.24"0.576 1.4"0.820 1.39"0.769 0.532"0.199

3

0.653"1.131

nd

3

113"23.6

3680"359

2.97"0.327

3 3 3

nd q nd

nd 5.37"4.37 2.74"0.76

3 3 3

57.0"14.1 14.3"4.30 169"105

3250"1570 1960"167 3120"1260

1,86"0.432 0.772"0.218 6.09"5.013

3 3 3 3

nd 2.77, nd nd nd

7.00"3.34 14.3"12.5 2.51"0.28 3.24"1.62

3 3 3 3

30.6"9.80 55.5"28.7 17.7"1 2.2 14.3"4.90

5470"2210 5450"564 2280"1290 4320"437

0.595"0.226 0.989"0.452 0.732"0.397 0.326"0.089

3 3

nd nd

2.89"0.80 2.58"0.63

3 3

19.3"14.4 28.2"10.8

3280"702 3730"1110

0.548"0.301 0.764"0.275

ECHINODERMAT, Echinoidea Anthocidaris crassispina Ovary Small intestine

3 2

1.24"0.42 q, 0.56

0.616"0.545 0.40, 0.34

60.8"1.32 62.3

3 2

74.8"11.9 32.9, nd

4100"2910 2780, 297

2.32"1.28 1.17, 1.13

PROCHORDATA, Ascidiacea Halocynthia reretzi Mantle Neural complex Ovary Stomach Branchial sac Endocarp Intestine

1 1 2 2 1 2 1

0.625 0.293 nd 2.11, 0.542 nd nd 2.33

nd nd 7.14 1.03, nd 2.61 nd nd

43.5

1 1 2 2 1 2 1

24.9 nd 26.1, 75.6 85.5, 17.5 13.2 nd nd

2220 116 4050, 5900 8150, 2040 147 626, 1000 882

1.11 0.641, 1.27 1.04, 0.850 0.89

MOLLUSCA, Cephalopoda Todarodes pacificus Liver Pancreas Ink sac Brain Tentacle Arm Eye Mantle Paroctopus conispadiceus Pedancle ganglion Cerebral ganglion Stellate ganglion Kidney Anterior salivary gland Posterior salivary gland Heart Gill (right) Branchial suspensor (right) Gastric caecum Liver Small intestine Branchial heart (right) Stomach Ovary

3 3 3 3 3 3

D y(DqL)

1.60

5880"742 7340"1340 6550"380 6440"767 4370"785 5170"906

0.573"0.023 0.906"0.169 0.500"0.143 0.453"0.007 0.59"0.140 0.466"0.042

2.73, 4.19 1.49, 0.794 1.20 2.26, 0.374 2.56, 1.99 1.98, 0.630 3.35, 0.348 0.689, 2.42

The contents and percentages are expressed as means"S.D., when the number of samples is 3 or more. Nd, not detectable; q, less than 1 pmol in 100 ml of the derivatized sample injected.

A. Tarui et al. / Comparative Biochemistry and Physiology Part B 134 (2003) 79–87

tamate in all the tissues examined is in contrast to the previous observation that animals containing higher amounts of NMDA also showed higher D y (DqL) ratio for aspartate. This may imply that Dglutamate is not metabolically linked to NMDG and does not have such physiological roles as expected and studied for D-aspartate. Acknowledgments This work was partly supported by a Grant-inAid for Scientific Research from Ministry of Education, Science, Sports, and Culture of Japan. We thank Mr Katsumasa Abe for his assistance in preparation of figures. References Aswad, D.W., 1984. Determination of D- and L-aspartate in amino acid mixtures by high-performance liquid chromatography after derivatization with a chiral adduct of o-phthaldialdehyde. Anal. Biochem. 137, 405–409. D’Aniello, A., Giuditta, A, 1977. Identification of D-aspartic acid in the brain of Octopus vulgaris. J. Neurochem. 29, 1053–1057.

87

D’Aniello, A., Giuditta, A., 1978. Presence of D-aspartate in squid axoplasm and in other regions of the cephalopod nervous system. J. Neurochem. 31, 1107–1108. Dawson, R.M.C., Elliott, D.C., Elliott, W.H., Jones, K.M., 1986. Data for biochemical research. 3rd ed. Oxford University Press, New York, pp. 463–464. Hersh, L.B., 1985. N-Methyl-L-glutamte synthase. Meth. Enzymol. 113, 36–42. Sato, M., Inoue, R, Kanno, N., Sato, Y, 1987. The occurrence of N-methyl-D-aspartic acid in muscle extracts of the blood shell, Scapharca broughtonii. Biochem. J. 241, 309–311. Shibata, K., Tarui, A., Todoroki, N., Kawamoto, S., Takahashi, S., Kera, Y, Yamada, R., 2001. Occurrence of N-methyl-Laspartate in bivalves and its distribution compared with that of N-methyl-D-aspartate and D,L-aspartate. Comp. Biochem. Physiol. 130B, 493–500. Todoroki, N., Shibata, K., Yamada, T., Kera, Y., Yamada, R., 1999. Determination of N-methyl-D-aspartate in tissues of bivalves by high-performance liquid chromatography. J. Chromatogr. 728B, 41–47. Yamada, T., Hasegawa, A., Matsumura, H., Uchiyama, T., Kera, Y., Yamada, R., 1996. Purification and properties of pig kidney D-aspartate oxidase and its utility in analysis of acidic D-amino acid. Bull. Nagaoka Univ. Technol. 18, 19–26. Watanabe, T, Shibata, K., Kera, Y., Yamada, R., 1998. Occurrence of free D-aspartate and aspartate racemase in the blood shell Scapharca broughtonii. Amino Acids 14, 353–360.