Kinetics of NAD reduction in the nucleus and the cytoplasm.

Abstract

The kinetics of the nuclear and cytoplasmic fluorescence response to glycolytic substrate were studied in ascites cells in culture (EL2 cells) and radiation giants (EL2G) maintained under a variety of conditions, using a beam-splitter supplemented microfluorimeter which allows fluorescence recording simultaneously with microelectrophoretic addition of substrate. A sequence of fluorescence pulses which resemble closely the curve of formation and disappearance of the enzyme-substrate complex were obtained upon repetitive additions of substrate. The pulses were analyzed in terms of peak fluorescence response (PFR), duration of steady state, halftime of fluorescence rise and decay (tin1/2off), number of consecutive cycles elicited, etc. There is a considerable parallelism in the kinetics of the nuclear and cytoplasmic fluorescence after addition of glycolytic substrate, over the whole time course of consecutive pulses. However in untreated, Amytal- or Rotenone-perfused cells the peak magnitudes of the cytoplasmic fluorescence are significantly lower and the cytoplasmic pulses are damped earlier than the nuclear upon repeated additions of substrate. In Amytal-grown EL2 cells there is a drop of PFR and a prolongation of t 1/2off in the nucleus and cytoplasm, which persists when the cells are transferred to an Amytal-free medium. However, if the cells are maintained for longer periods in Amytal, the nuclear fluorescence tends to return to control level, while the cytoplasmic pulses remain small and are easily damped. In T3- or Amytal + T3 -grown EL2 cells the cytoplasmic fluorescence instead of dropping like in the controls, follows the nuclear level over the whole time course of repeated pulses, and can even exceed the nuclear. Comparable phenomena are observed in T3 -and Insulin-grown radiation giants When the amount of substrate is varied, starting from levels which can barely elicit a response the magnitude of the fluorescence response (integrated fluorescence pulse) in the cytoplasm and nucleus follows a sigmoid curve which can be interpreted as a function of allosteric enzymes.