Determination of D-aspartate N-methyltransferase activity in the starfish by direct analysis of N-methyl-D-aspartate with high-performance liquid chromatography |
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Authors: | Shibata Kimihiko Sugaya Noriko Ono Wakana Abe Katsumasa Takahashi Shouji Kera Yoshio |
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Affiliation: | Department of Chemistry and Biochemistry, Fukushima National College of Technology, 30 Nagao, Iwaki, Fukushima 970-8034, Japan. shibata@fukushima-nct.ac.jp |
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Abstract: | We describe a method for the detection and quantification of D-aspartate N-methyltransferase activity. The enzyme catalyzes the S-adenosyl-L-methionine-dependent N-methylation of D-aspartate to form N-methyl-D-aspartate (NMDA). NMDA is detected directly by high-performance liquid chromatography (HPLC) of their (+)- and/or (-)-1-(9-fluorenyl)ethyl chloroformate fluorescent derivatives. The NMDA production in the assay mixture is linearly proportional to the incubation time and the amount of tissue homogenate. Using a 10 min incubation time, the method allows detection of the enzyme activity below 10 fmol/min. It can be used to analyze kinetic behavior and to quantify the enzyme from a wide variety of organisms. |
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