Action of acriflavine on the growth and mutation in yeast II. Kinetic study on the effect of glutamate on the respiration-deficient mutation induction

Abstract

Enrichment with glutamate (G) of an acriflavine-containing medium increased the maximum frequency of a respiration-deficient mutant (RD max) in yeast culture. The RD max value was directly related to the mutation rate calculated in the logarithmic growth phase and was shown by = k·RD max, k being about 0.0042 independent of temperature. Effects of concentrations of glutamate ([G]) and acriflavine ([AC]) on the mutation rate were studied at various temperatures, kinetically analogous with enzyme kinetics. Citing changes of the “Michaelis constant”, evidence is presented which strongly supports the theory that two molecules of AC and one molecule of G are reacting in the order of (1) G-AC-AC and (2) AC-G-AC to induce the mutation. The change induced by temperature in the “Michaelis constant” of the glutamate reaction in (1) indicated that the glutamate reaction is an endothermic synthetic reaction in which the heat change is about 18 kcal. The efficiency of AC, κ, in increasing π was increased by G in accordance with the Michaelis-type equation at constant temperature. The change of the “Michaelis constant” ( K k ) due to temperature suggested that the efficiency of AC is increased by an endothermic glutamate reaction of which the heat change is about 22 kcal. The value of κ, which varies according to temperature, was determined at an early phase of culture, up to 6–8 h at 29°, in the presence of AC. Once the κ was determined, withdrawal of AC or shifting-down of incubation temperature did not affect the κ value. However, the absolute value of π was increased markedly by the lowering of the growth rate due to shifting-down of temperature. This cannot be accounted for by considering that the dilution of non-impaired ϱ by cell growth is a main part of the RD induction mechanism. A new hypothesis is presented that the action, on ϱ, of a temperature-dependent mutator gene or of very inaccurate polymerase is affected by glutamate and acriflavine.