Establishment and application of activity determining methods for different subtypes of xanthine oxidoreductase
10.16438/j.0513-4870.2023-0389
- VernacularTitle:黄嘌呤氧化还原酶不同亚型活性测定方法的建立与应用
- Author:
Dong-ting CHEN
1
;
Jin-ying TIAN
1
;
Xue-chen LI
1
;
Jiang LI
1
;
Fei YE
1
Author Information
1. Beijing Key Laboratory of New Drug Mechanisms and Pharmacological Evaluation Study, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College, Beijing 100050, China; Diabetes Research Center of Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
- Publication Type:Research Article
- Keywords:
xanthine oxidoreductase;
xanthine dehydrogenase;
xanthine oxidase;
enzyme activity determination;
enzyme kinetics;
inhibitor
- From:
Acta Pharmaceutica Sinica
2023;58(10):3016-3023
- CountryChina
- Language:Chinese
-
Abstract:
Xanthine oxidoreductase (XOR), the key enzyme catalyzing purine to produce uric acid, including two subtypes, xanthine dehydrogenase (XDH) and xanthine oxidase (XO), respectively, in vivo. Usually, XDH and XO can transform to each other. In this study, based on the principle that the subtype XO or XDH uses different electron acceptors, the methods for the measuring the activities of bovine milk XOR (pure enzyme) and its subtypes were established. The optimal concentrations of substrate xanthine (50 μmol·L-1) and electron acceptor NAD+ (50 μmol·L-1), pH value (7.80) were investigated. The ranges of the XOR, XO, XDH activity which could be determined were 0.97-17.5 U·L-1, 1-9 U·L-1, and 66-1 191 mU·L-1, respectively. Furthermore, the methods for determining the activities of XOR and its subtypes in mouse liver were established. The preparation of liver samples, the optimal concentrations of xanthine (100 μmol·L-1) and NAD+ (100 μmol·L-1) were researched. And the activity ranges of XOR, XO and XDH in mouse liver which could be determined were 0.67-3.98, 0.19-1.08, and 0.52-3.55 U·gprot-1, respectively. With the methods above, the effects of classic XOR inhibitor allopurinal (Allo) on XOR, XO and XDH from both milk and mouse liver were determined. All animal experiments have been approved by the Animal Experimental Center, Institute of Materia Medica, Chinese Academy of Medical Science and Peking Union Medical College (00003346). This study established new methods for the determination of XOR and its subtypes activity in pure enzyme system and in mouse liver, respectively, which were accurate and convenient. It laid the experimental foundation for exploring the different pathophysiological effects of XOR in the body and developing new XOR inhibitors.
