TY - JOUR
T1 - A colorimetric assay method for measuring D-glutamate cyclase activity
AU - Katane, Masumi
AU - Motoda, Risa
AU - Ariyoshi, Makoto
AU - Tateishi, Shuhei
AU - Nakayama, Kazuki
AU - Saitoh, Yasuaki
AU - Miyamoto, Tetsuya
AU - Sekine, Masae
AU - Mita, Masashi
AU - Hamase, Kenji
AU - Matoba, Satoaki
AU - Sakai-Kato, Kumiko
AU - Homma, Hiroshi
N1 - Funding Information:
This work was supported by JSPS KAKENHI [grant number: JP18K06117 ] (to MK) and the Research Foundation for Pharmaceutical Sciences (to MK).
Publisher Copyright:
© 2020 Elsevier Inc.
Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.
PY - 2020/9/15
Y1 - 2020/9/15
N2 - In mammals, metabolism of free D-glutamate is regulated by D-glutamate cyclase (DGLUCY), which reversibly converts D-glutamate to 5-oxo-D-proline and H2O. Metabolism of these D-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-D-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-D-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet–visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of D-glutamate and 5-oxo-D-proline, and cardiac function.
AB - In mammals, metabolism of free D-glutamate is regulated by D-glutamate cyclase (DGLUCY), which reversibly converts D-glutamate to 5-oxo-D-proline and H2O. Metabolism of these D-amino acids by DGLUCY is thought to regulate cardiac function. In this study, we established a simple, accurate, and sensitive colorimetric assay method for measuring DGLUCY activity. To this end, we optimized experimental procedures for derivatizing 5-oxo-D-proline with 2-nitrophenylhydrazine hydrochloride. 5-Oxo-D-proline was derivatized with 2-nitrophenylhydrazine hydrochloride in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide as a catalyst to generate the acid hydrazides, whose levels were then determined using a colorimetric method. Under optimized conditions, we examined the sensitivity and accuracy of the colorimetric method and compared our technique with other methods by high-performance liquid chromatography with ultraviolet–visible or fluorescence detection. Moreover, we assessed the suitability of this colorimetric method for measuring DGLUCY activity in biological samples. Our colorimetric method could determine DGLUCY activity with adequate validity and reliability. This method will help to elucidate the relationship among DGLUCY activity, the physiological and pathological roles of D-glutamate and 5-oxo-D-proline, and cardiac function.
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U2 - 10.1016/j.ab.2020.113838
DO - 10.1016/j.ab.2020.113838
M3 - Article
C2 - 32702438
AN - SCOPUS:85088642156
VL - 605
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
M1 - 113838
ER -