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

Determination of d-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-d-aspartate with high-performance liquid chromatography

“…The method used in the present study allows clear separation of NMDA, NMLA, NMDG and NMLG using (+)-and (−)-FLEC, as well as those described in our previous study [2][3][4]. Therefore, the HPLC assay procedure developed here seems to be compatible with detailed kinetics studies of d-aspartate N-methyltransferase in living organisms.…”

“…NMDA and NMLA were identified as described below. NMLG was identified by the behaviors of the (+)-FLEC and (−)-FLEC derivatives of NMLG on HPLC chromatograms, as described in our previous study [4] (data not shown). Since radial nerve cord had the highest concentrations of NMDA and NMLA not only among the starfish tissues examined but also in various tissues of invertebrates so far reported [2,3], we decided to measure d-aspartate N-methyltransferase activity with radial nerve code as the enzyme source.…”

“…This situation prompted us to develop a simple, direct and reliable assay method for d-aspartate N-methyltransferase activity in animal tissues. Our method reported in the previous studies using (+)-FLEC is able to separate of NMDA from the other N-methylamino acids [3,4]. On the basis of the method [2], we report here the development of a direct and reliable assay method for d-aspartate N-methyltransferase activity using a simple HPLC system, and its application in starfish tissues, in which we found a large amount of NMDA together with other acidic N-methylamino acids.…”

“…One of them is an enantiomer of NMDA, N-methyl-l-aspartate (NMLA) that was found only in two bivalve species [3]. Two of them found in S. broughtonii, S. sub- crenata and several other invertebrates are N-methyl-d-glutamate (NMDG) and N-methyl-l-glutamate (NMLG) [4]. The discovery of the natural occurrence of these N-methyl-d,l-amino acids has suggest the possibility that these compounds were widely distributed in many other living organisms with some significant functions.…”

“…1). In view of the significance of the discovery, more supporting evidence should be provided for this enzyme system, since the employed assay method leaves some uncertainty: the method detects methylamine and not NMDA itself [5,[7][8][9], and uses d-aspartate oxidase from beef kidney, which may oxidize NMDG, as the oxidase from pig kidney does [4,10], and/or other unknown compound(s) present in the tissues to form methylamine. Thus it is not certain, whether all methylamine detected comes from NMDA.…”