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A colorimetric assay method for measuring d-glutamate cyclase activity.
Katane, Masumi; Motoda, Risa; Ariyoshi, Makoto; Tateishi, Shuhei; Nakayama, Kazuki; Saitoh, Yasuaki; Miyamoto, Tetsuya; Sekine, Masae; Mita, Masashi; Hamase, Kenji; Matoba, Satoaki; Sakai-Kato, Kumiko; Homma, Hiroshi.
Afiliação
  • Katane M; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Motoda R; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Ariyoshi M; Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan.
  • Tateishi S; Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan.
  • Nakayama K; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Saitoh Y; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Miyamoto T; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Sekine M; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Mita M; Shiseido Co., Ltd, 1-1-16 Higashi-shimbashi, Minato-ku, Tokyo, 105-0021, Japan.
  • Hamase K; Department of Drug Discovery and Evolution, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan.
  • Matoba S; Department of Cardiovascular Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto, 602-8566, Japan.
  • Sakai-Kato K; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan.
  • Homma H; Laboratory of Biomolecular Sciences, Graduate School of Pharmaceutical Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo, 108-8641, Japan. Electronic address: hommah@pharm.kitasato-u.ac.jp.
Anal Biochem ; 605: 113838, 2020 09 15.
Article em En | MEDLINE | ID: mdl-32702438
ABSTRACT
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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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Colorimetria / Hidroliases Tipo de estudo: Diagnostic_studies Limite: Animals Idioma: En Ano de publicação: 2020 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Colorimetria / Hidroliases Tipo de estudo: Diagnostic_studies Limite: Animals Idioma: En Ano de publicação: 2020 Tipo de documento: Article