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ULTRAMICRO METHODS FOR DETERMINING ENZYME ACTIVITIES AND SUBSTRATES USING COMBINED BIOLUMINESCENT ASSAY WITH ENZYMATIC CYCLING

使用生物发光测定与酶循环相结合测定酶活性和底物的超微方法

基本信息

批准号：
62870009
负责人：
ENDOU Hitoshi
金额：
$ 4.29万
依托单位：
UNIVERSITY OF TOKYO
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

To investigate metabolic and pharmacological properties in individual cells constituting heterogenous organs or tissues, ultramicro analysis sensitive enough to be applicable to a single cell should be required. In this project, combined bioluminescent assays with enzymatic cycling methods gave been established.1) Automatic analysis of bioluminescence. An automatic analysising system has been established by comining a luminometer (LKB-1251) and personal computer (PC 8201) with a recorder or a printer.2) Micromethod of ATP determination. Extraction of cellular ATP by 10 % trichloroacetic acid has been found to be the best. A tiny amount of ATP less than 10^<-12> mol could be assayed with firefly luciferin and luciferase using the luminometer. By this analytical procedrue, substrate specificity to maintain cellular ATP was clarified in various mouse nephron sehments.3) Ultramicro analyses of of Na^+,K^+-ATPase activities and fmolar orders of ammonia. Using HADH: FMN oxidoreductase and luciferase, Na^+,K^+-ATPase activity could be quantified by determining pyruvate coverted from phosphoeno lpyruvate in the presence of ADP (a metabolyte of ATP splitted by ATPase) and pyruvate kinase. When enzymatic cycling of NAD was compined with bioluminescent assay, 10^<-16> mol ADP could be determined. This ultramicro method can be applied for the determination of ATPase activity in a single cell. Similarly, fmol ammonia could be analized. These methods were successfully applied for the study of intranephron ammoniagenesis.4) Ultramicro assay of glucose. Tiny amounts of glucose (less than 10^<-15> mol) could be quantified using combined bioluminesxent assay of NADPH with NADP/NADPH enzymatic cycling method. This analytical procedure has been applied to intrenephron gluconeogenesis study.5) Real time analysis of superoxide. Using luminol, superoxide stimulated by phorbol ester in isolated glomeruli could be successfully monitored.

为了研究构成异质器官或组织的单个细胞的代谢和药理学特性，需要进行灵敏度足以适用于单个细胞的超微分析。本课题建立了生物发光与酶循环联用技术。1）生物发光的自动分析。用LKB-1251型光度计和PC-8201型微机，配以记录仪或打印机，建立了自动分析系统。用10%三氯乙酸提取细胞ATP效果最好.用<-12>荧光素酶和萤火虫荧光素酶可测定小于10 μ mol的微量ATP。3）Na^+，K^+-ATP酶活性和氨的摩尔级数的超微分析。使用HADH：FMN氧化还原酶和荧光素酶、Na^+，K^+-ATP酶活性可通过测定在ADP（ATP酶分解ATP的代谢产物）和丙酮酸激酶存在下磷酸烯醇丙酮酸转化为丙酮酸来定量。将酶促NAD循环与生物发光法结合，可测定10 μ <-16>mol ADP。该方法可用于单细胞ATP酶活性的测定。类似地，可以分析fmol氨。这些方法成功地应用于肾内氨合成的研究。4）葡萄糖的超微量测定。微量的葡萄糖（小于10 μ <-15>mol）可以使用NADPH与NADP/NADPH酶循环方法的组合生物发光测定来定量。该方法已用于肾单位内脂质过氧化产物的研究。5）超氧化物的真实的实时分析。使用鲁米诺，超氧化物刺激佛波酯在离体肾小球可以成功地监测。