In vivo studies of pyridine nucleotide metabolism in Escherichia coli and Saccharomyces cerevisiae by carbon-13 NMR spectroscopy.

Pyridine nucleotide metabolism has been studied in vivo in a prokaryotic (Escherichia coli) and a eukaryotic (Saccharomyces cerevisiae) system cultured in a medium containing carbon-13-labeled nicotinic acid, followed by NMR detection of the labeled organisms. Chemical exchange between oxidized and reduced nucleotides is found to be sufficiently slow on the NMR time scale to permit the observation of separate resonances corresponding to each redox state. The possibility of significant exchange broadening of reduced pyridine nucleotide resonances under some conditions was further evaluated based on comparative NMR studies utilizing organisms cultured in the presence of either [2-13C]nicotinate or [5-13C]nicotinate. Based on these experiments, it was concluded that broadening as a consequence of intermediate exchange is not significant. Although it was initially anticipated that the carbon-13 resonances arising from the di- and triphosphopyridine nucleotide pools could not be distinguished, the absence of observable resonances corresponding to reduced nucleotides in oxygenated yeast and E. coli cells suggests that the NMR method is fairly specific for determining the redox status of the diphosphopyridine nucleotide pool. Studies of the effects of a variety of perturbations including variation of the oxygen supply, addition of ethanol, and addition of the oxidative phosphorylation uncoupler dinitrophenol have been carried out. Dramatic differences in the response of the catabolic reduction charge, CRC = [NADH]/[NADH] + [NAD+], between the yeast and E. coli cells are observed. The CRC values for the yeast undergo large changes in response to these perturbations which are not observed for the bacterial cells.