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

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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.