Damped Sinusoidal Oscillations of Cytoplasmic Reduced Pyridine Nucleotide in Yeast Cells.

The most obvious periodicities of biological systems are the "clock phenomena."1 An example of periodic fluctuations at the biochemical level is provided by the luminescence of Gonyaulax.2 In this case, at least the intracellular localization of the biological system involved is known,1 but the nature of the oscillator driving such clocks is unknown at either the physiological or the biochemical level. While the transition from aerobiosis to anaerobiosis causes changes of cytochrome oxidation-reduction that are monotonic,4 the concentration of intracellular reduced pyridine nucleotide (RPN) in yeast was observed to fluctuate widely in a cyclic fashion on the first occasion that direct optical method was applied to intact cell suspensions.4' Various explanations for these phenomena were considered, involving the possibility of binding of the reduced4 or the oxidized6' form of pyridine nucleotide, particularly to glyceraldehyde-3-phosphate dehydrogenase. In 1957, Duysens and Amesz,8 using fluorescence detection of reduced pyridine in yeast cell suspensions, observed a related and more extensive fluctuation in the fluorescence intensity. Estabrook et al.,9 also employing the fluorometric method, observed cyclic fluctuations of cytoplasmic reduced pyridine nucleotide in suspensions of E. chili. In experiments with an inositol-requiring strain of Saccharomyces carlsbergensis (ATCC-4228), the generation of damped sinusoidal wavetrain of over 12 full cycles of oscillations was observed in 1963.9' 10 These results were confirmed10 through the use of the direct optical method; the oscillation was found to involve changes in the oxidation-reduction state of RPN rather than changes of its binding. Metabolite assays11 of the cell suspension of the oscillation indicated fluctuation of the glycolytic intermediates showing a crossover point at phosphofructokinase. The assays further show the fluctuations in fructose diphosphate concentration to be out of phase with glucose-6-phosphate and fructose-6-phosphate, leading to a simplified mechanism involving phosphofructokinase as the oscillatory element in the enzymatic sequence.12 In 1955, Wilson and Calvinl2a found brief oscillations of diphosphoglycerate and ribulose diphosphate upon illumination of Scenedesmus. An example of such oscillation is provided by one of the first recordings of reduced pyridine nucleotide kinetics in yeast cells (Fig. 1) by the double beam spectrophotometer.4' 335 mti and 350 m~u were employed as measuring and reference wavelengths, respectively, in view of the slight shift of the reduced pyridine nucleotide band of yeast cells to shorter wavelengths. Addition of 33 mM sucrose (which is rapidly converted to glucose in the yeast cells by invertase) causes the reduced pyridine nucleotide concentration to rise abruptly to a peak in 10 see as indicated by the downward deflection of the trace. An approximately full cycle of oscillation ensues thereafter in which a second peak is reached in the course of slightly over a minute. Inhibitor studies identified the oscillation with the glycolytic rather than the oxidative enzymes and with glyceraldehyde-3-phosphate dehydrogenase. This cyclic response has been observed in ascites tumor cells13 and more recently in