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

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，需要足够敏感的适用于单个细胞的超微分析。本项目建立了生物发光与酶循环相结合的检测方法。1)生物发光自动分析。用LKB-1251光度计和PC 8201个人计算机（带有记录仪或打印机）组成了一个自动分析系统。2)微量法测定ATP。用10%三氯乙酸提取细胞ATP的效果最好。荧光素和荧光素酶可以用荧光计测定小于10^<-12> mol的微量ATP。通过这种分析方法，阐明了在不同小鼠肾元片段中维持细胞ATP的底物特异性。3) Na^+、K^+- atp酶活性和氨摩尔阶的超微分析。利用HADH: FMN氧化还原酶和荧光素酶，可以通过测定磷酸基丙酮酸在ADP （ATP被ATP酶分裂的代谢产物）和丙酮酸激酶存在下转化的丙酮酸来定量Na^+、K^+-ATP酶活性。当NAD酶循环与生物发光法结合时，可以测定出10^<-16> mol ADP。该方法可用于单细胞中atp酶活性的测定。同样，fmol氨也可以分析。这些方法已成功地应用于肾内氨法的研究。4)葡萄糖的微量测定。利用NADP/NADPH酶循环法联合生物发光法可以定量测定微量葡萄糖（小于10^<-15> mol）。本分析方法已应用于干扰素糖异生的研究。5)超氧化物实时分析。利用鲁米诺，可以成功地监测离体肾小球中佛博尔酯刺激的超氧化物。

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.