黄嘌呤氧化还原酶不同亚型活性测定方法的建立与应用  

Establishment and application of activity determining methods for different subtypes of xanthine oxidoreductase

作　　者：陈冬婷 田金英[1,2] 李雪晨 李江[1,2] 叶菲 CHEN Dong-ting;TIAN Jin-ying;LI Xue-chen;LI Jiang;YE Fei(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)

机构地区：[1]中国医学科学院,北京协和医学院药物研究所,北京新药机制与药理评价研究重点实验室,北京100050 [2]中国医学科学院糖尿病研究中心,北京100050

出　　处：《药学学报》2023年第10期3016-3023,共8页Acta Pharmaceutica Sinica

基　　金：中国医学科学院医学与健康科技创新工程(2021-I2M-1-029,2022-I2M-1-020);中国药学会-以岭生物医药创新基金(CPA-B04-ZC-2021-005);国家自然科学基金青年科学基金项目(22107121)。

摘　　要：黄嘌呤氧化还原酶(xanthine oxidoreductase,XOR)是体内催化嘌呤生成尿酸的关键酶,以两种可以相互转化的亚型存在,分别是黄嘌呤脱氢酶(xanthine dehydrogenase,XDH)和黄嘌呤氧化酶(xanthine oxidase,XO)。本研究首先通过两亚型酶在催化过程中使用电子受体不同的原理,在牛奶来源纯酶中建立了XOR及亚型XO、XDH的活性测定方法,明确了测定体系中最适底物xanthine浓度为50μmol·L^(-1),最适电子受体烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)浓度为50μmol·L^(-1),最适pH值为7.80,XOR、XO、XDH的可测定范围分别为0.97~17.5 U·L^(-1)、1~9 U·L^(-1)、66~1191 mU·L^(-1)。随后在纯酶体系测定方法的基础上,建立了小鼠肝组织XOR及亚型XO、XDH的活性测定方法,明确了组织制样方法、测定体系中最适xanthine浓度为100μmol·L^(-1)、最适NAD+浓度为100μmol·L^(-1),XOR、XO、XDH的可测定范围分别为0.67~3.98、0.19~1.08、0.52~3.55 U·gprot^(-1)。应用已建立的方法,测定了经典的XOR抑制剂别嘌呤醇(allopurinal,Allo)对牛奶及小鼠肝组织来源XOR、XO及XDH活性的抑制作用。所有动物实验经过中国医学科学院药物研究所实验动物管理与动物福利委员会审核并批准(00003346),符合实验动物伦理相关规范。本研究分别建立了牛奶来源纯酶体系中及小鼠肝脏中XOR及其亚型XO、XDH的活性测定方法,该方法准确、便捷,对探究XOR在机体中不同的病理生理作用、研发新型XOR抑制剂等奠定了实验基础。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(50μmol-L^(-1))and electron acceptor NAD^(+)(50μmol-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 66-1191 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(100μmol-L^(-1))and NAD^(+)(100μmol-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.

关 键 词：黄嘌呤氧化还原酶 黄嘌呤脱氢酶 黄嘌呤氧化酶 酶活性测定 酶动力学 抑制剂 

分 类 号：R965.2[医药卫生—药理学]