Some Characteristics of the Fluorescence Lifetime of Reduced Pyridine Nucleotides in Isolated Mitochondria, Isolated Hepatocytes, and Perfused Rat Liver In Situ

作者：Masayoshi Wakita, Goro Nishimura, Mamoru Tamura · 发表于：The Journal of Biochemistry · 年份：1995 · DOI：10.1093/oxfordjournals.jbchem.a125001 · 被引用次数：110 · 研究领域：Mitochondrial Function and Pathology、Birth, Development, and Health、Metabolism and Genetic Disorders

By extensively examining the experimental conditions for time-resolved spectrophotometry of non-transparent light scattering systems, we demonstrated the feasibility of quantitative analysis of both the fluorescence lifetime and intensity of reduced pyridine nucleotides in living tissues, suspensions of isolated liver mitochondria, and hepatocytes, as well as hemoglobin-free perfused rat liver being used systematically for measurements. The fluorescence decay was analyzed by the maximum likelihood method with a 4-component decay model. The lifetime of NADH observed in mitochondria (mean: 2.8 +/- 0.2 ns) was much longer than that of the free form in an aqueous solution (mean: 0.43 +/- 0.01 ns), and it was characterized as a protein-bound form. The lifetime was not affected by either aerobic or anaerobic conditions nor by the energy state, though the intensity changed markedly. The decay curves of isolated hepatocytes under normal aerobic conditions were the same as those of isolated mitochondria, though cytosolic NADH and NADPH were superimposed. Under the conditions of "unphysiological" acidosis, the mean lifetime became about 1.5 times longer than that under normal conditions. With perfused liver, the relative contributions of cytosolic NADH and NADPH were determined by infusing lactate and tert-butylhydroperoxide. Cytosolic NADH did not contribute to the overall fluorescence of pyridine nucleotides. In contrast, about 70% of the total fluorescence intensity was due to cytos...