Localization and Kinetics of Reduced Pyridine Nucleotide in Living Cells by Microfluorometry

Abstract

On the basis of early studies of the blue fluorescence of living cells and tissues before chemical treatment,l Sjiistrand (1) suggested its association with the mitochondrial bodies. Microspectroscopic observations of prepared tissue sections revealed emission bands of the fluorescent material of axons (1) and acidtreated groups of kidney cells;2 critical evaluations of available spectrograms of purified materials lead to the identification of thiamin and riboflavin, respectively. Although some of the kidney sections, before acid treatment, showed fluorescence bands in the spectrograms that are now regarded as suggestive of reduced pyridine nucleotide, the fluorescence of which was first observed by Warburg (2), insufficient data were available at that time to consider reduced pyridine nucleotide as a possible cause of the tissue fluorescence. Recent studies by Boyer and Theorell (3) and Duysens and Kronenberg (4) on alcohol dehydrogenase show clearly the great enhancement of DPNH fluorescence that is caused by a binding of the coenzyme to the enzyme surface. Furthermore, Duysens and Amesz (5) demonstrate that the intact yeast cell shows a fluorescence characteristic of bound reduced pyridine nucleotide. In more recent experiments, it has been found that intramitochondrial reduced pyridine nucleotide also exhibits the same characteristic fluorescence, calling attention to the possibility of a close relationship between this effect and the blue fluorescence of living cells and tissues (6). The fluorometric result agrees with the spectrophotometrically determined large RPN3 content of mitochondria (7). Furthermore, its binding to a mitochondrial component has been suggested by kinetic studies (7). More recent data show that the fluorescence of intact muscle diminishes upon electrically induced contraction, in agreement with the spectrophotometrically observed oxidation of RPN (8). Thus, there is good evidence that a considerable amount of tissue fluorescence is due to this component. To study the fluorescence of mitochondrial RPN independently of that of the cytoplasm, it has been desirable to develop a microfluorometric method, which, in conjunction with suitable biological materials showing isolation of the mitochondrial bodies, could be used to investigate cytoplasmic-mitochondrial interactions and also to permit the assay of RPN localized in different