On the effect of dinitrophenol on carbohydrate activation in higher plant tissues. — Previous investigations on the effects of 2,4 dinitrophenol (DNP) on carbohydrate metabolism in isolated pea internodes and in yeast showed that the increased rate of glycolysis induced by the uncoupler corresponds to an increased rate of the conversion of free hexoses and polysaccarides to hexose phosphates. In yeast about 30% of the radioactivity supplied and taken up as 14C labelled glucose, and 20% of that supplied and taken up as glycerol is recovered as soluble sugar and glycogen; this phenomenon is almost completely suppressed by 10-4M DNP. This suggested that a mechanism involving kinase enzymes, on one hand, and phosphatases, on the other, is mediating the interconversion of phosphorylathed and free sugars, and that the apparent increase of hexose phosphorylation observed in the presence of DNP might depend on a decreased rate of phosphatase mediate reactions, consequent to the decrease of phosphorylated sugars level in the cell. The experiments here reported were planned to test the validity of this hypothesis in the case of higher plant tissues. Material used in these experiments were segments from the growing part of the third internode isolated from 7 day old, etiolated pea seedlings, and carrot root diks (0,7 mm thick, 7 mm diameter) preincubated for 24 hours in aerated distilled water. Both of these materials show an active, steady respiration and some growth activity, so that they may be taken as representing a condition close enough to that of the generally physiologically active higher plant tissues. The reversibility of the hexose phosphate-free sugar interconversion process was tested by feeding 10-3M 1-C14 labeled glycerol, and measuring after 150 minutes the amount of radioactivity incorporated into CO2, soluble sugars, organic acids and proteins. The results of these experiments are summarized in table I and II. Glycerol metabolism as well as its response to DNP appears very similar in the two material used. In both cases, glycerol uptake and incorporation into organic acids and amino acids is almost insensitive to DNP. In contrast large differences are observed for the free sugar fraction. In the absence of the uncoupler, a consistent amount of the radioactivity fed as glycerol is found in this fraction. It appears reasonable to assume that the glycerol-sugar interconversion comprehends, as intermediate steps, glycerol-P, fructose di-P (or sedoeptulose di-P) and hexose-6-P. If this is true, the observed data implicate that a continuous interconversion occurs, in the cell, between sugar phosphates and free sugars and vice-versa, one reaction direction involving the activity of phosphatases, and the other one that of kinases. The true rate of this interconversion process is probably much larger than indicated by the radioactivity found in free sugars: as a considerable part of the triose-P transormed into sugars must immediately re-enter the descending flux of glycolysis. This view finds some support in the fact that DNP almost completely inhibits the incorporation of radioactivity in the free sugar fraction. It has been previously observed that DNP very markedly decreases the level of hexose mono- and di-phosphates and of triose-phosphates in the pea stem tissues. If phosphatases acting on fructose di-phosphate and on hexose-6-P are not saturated by their substrates, a decrease of the rate of free hexose synthesis from sugar phosphates should be expected. The present results are thus consistent with the hypothesis that hexose phosphates and free sugars in the cell are continuously interconverted by the simultaneous action of phosphatases and kinases; and that the effect of DNP, and thus of any physiological conditions decreasing the ATP/ADP ratio in accelerating free hexose utilizations is at least in part due to a decreased rate of the reactions catalized by fructose diphosphate and hexose-6-P phosphatases. The reversibility of the kinase-phosphatase system would thus represent a crucial link in the mechanism by which the rate of carbohydrate activation and breackdown is controlled by the rate of utilization of high-energy phosphate bonds.