Azione Del 2,4- Dinitrofenolo E Della Tensione Di Ossigeno Sull'Attivazione Respiratoria Di Fettine Di Tubero Di Patata

Abstract

The effects of oxygen and of dinitrophenol on the respiratory activation of potato tuber slices. — It is well known that thin slices from storage organs show, in the first period following preparation and washing, a transition from a low to a high level of metabolic activity. In the potato tuber discs (and similar materials) this transition is characterized by: a) a several fold increase of oxygen uptake; b) an increase of the capacity of dissimilating to CO₂ the carbon 1 of glucose; c) a decrease of the sensibility of oxygen uptake to the uncoupler 2,4 dinitrophenol (DNP); d) an increase of the capacity of ion and sugar uptake; e) the development of marked capacity of protein synthesis; f) and increase of the activity (as determined in homogenates) of several enzymes involved in the directs oxidation of glucose-6-P or — possibly — in the reoxidation of the TPN reduced by the pentose phosphate pathway reactions. The two latter responses appear with some delay respect the activation of oxygen uptake, which suggested (Marrè et al.) that they are probably rather a consequence than a primary cause of the activation of respiration.

The present researches are aimed to a better understanding of the role played by the activity of oxidative enzymes and by high energy phosphate acceptor availability during the activation of metabolism in potato tuber slices.

The importance of these factors was investigated by measuring the QO₂ under different O₂ pressure conditions, and by following the respiratory effects induced by DNP supplied at various concentration and at various incubation times. The results obtained can be summarized as follows:

I. - Changes induced by varying O₂ pressure.

a) Lowering O₂ pressure from 20% (air) to 5% does not affect the QO₂ of freshly prepared slices, showing a low respiration rate, while it consistently depresses (by 40–50%) the QO₂ of slices preincubated in air for periods longer than 8 hours, in which the process of respiratory activation is already well developped. At any stage of aging of the slices, no difference in QO₂ is observed when air is substituted with pure oxygen. The rate of respiratory activation of the discs is highly sensitive to O₂ partial pressure during incubation. At 20% O₂ (air) the system involved in activation is practically satured, no further increase of the activation rate being observed at higher O₂ (100%). With 5% O₂, however, the rate of activation (measured after bringing again the slices in 20% oxygen) appears markedly depressed.

b) The increase, upon incubation, of the dissimilation of carbon 1 of glucose to CO₂ is also strongly depressed by 5% O₂. This suggests that the progressive increase of the capacity of the oxidative system during aging makes limiting — at a given moment — the rate of diffusion of oxygen in the tissues.

c) The increase during incubation of the capacity of active uptake of solutes such as the Rb+ ion and glucose is not significantly depressed by the low (5%) oxygen pressures. This seems to be in relation with the early development of this aspect of the activation process during the first period of activation, when the respiratory rate is low and lowering O₂ pressure does not affect QO₂. On the other hand, the lack of sensibility of ion and glucose uptake to low oxygen in the aged slices could indicate that the high energy phoshate level required for this process is lower than that for other processes, such as protein synthesis.

d) Protein synthesis activation (measured as radioactivity incorporation into proteins in slices fed with glucose-6-C¹⁴) is severely inhibited (by about 60%) by 5% O₂. This suggests a close correlation between respiratory activity and protein synthesis, on one hand, and between protein synthesis and the activation process occurring during aging, on the other.

II. - Changes induced by DNP at different O₂ pressures.

a) As already shown in other laboratories, DNP at an appropriate concentration (in our case around 3 × 10^-5M, at pH 5) markedly stimulates O₂ uptake in the fresh slices, and this effect gradually disappears, parallely with the respiratory activation, when DNP is supplied to slices preincubated in distilled water for increasing periods of time. When slices were incubated from the beginning with the DNP concentration giving maximal respiratory response (3 × 10^-5M at pH 5), the respiratory increase due the incubation was completely suppressed and the control slices incubated in water reached, after a few hours (3–5), QO₂ values, higher than those of the DNP treated slices. With DNP concentration between 10^-5M and 3 × 10^-5M the initial rate of respiration was still higher than in the controls; this positive difference very slowly disappeared with the increase of incubation time, until, after about 36 h (when respiratory activation in the controls approached its maximum) the two curves became practically identical.

A large fraction of the DNP induced increase of QO₂ can be readily reversed by washing the slices and removing the inhibitor. When this is done, the rate of respiration observed for the slices preincubated (up to 10 hours) in DNP concentration giving maximal positive respiratory response falled down to values close to those of the fresh slices; while in the case of lower DNP concentration the level of respiration is almost unaffected by washing off the inhibitor. These results confirm the hypothesis that lack of phosphate acceptors is strongly limiting respiration in the « fresh » slices, and that this type of control is slowly losing its importance during the « aging » process: which appears to be a consequence of both the increased activity of ATP utilizing systems and the appearance of some physiological (facultative) uncoupling mechanism.

b) Immediately after preparation, when O₂ uptake is very low, the respiration of both the slices in 20% and in 5% O₂ is stimulated by DNP at approximately the same extent. However, when slices incubated in air for 7–8 hours, (still showing a significant, although reduced, respiratory response to DNP), are brought to 5% O₂, the effect of DNP is completely suppressed. Conversely, the QO₂ of slices incubated for the same period with 5% O₂ (which inhibits respiratory activation, see I), do not respond to DNP when the O₂ pressure is mainteined low, while they show a clear response when O₂ in the gas phase is rised to 20%. The fact that in the « partially aged » (that is, incubated for a 7–9 h. period) slices lowering O₂ to 5% simultaneously inhibits O₂ uptake and completely suppresses the DNP effect, suggests that the respiratory fraction sensitive to O₂ pressure is involved in (mitochondrial) oxidative phosphorylation. The simplest interpretation is in fact that in low O₂ the ADP level rises, and that oxidative activity, instead of phosphate acceptor level, becomes the main factor limiting respiration.

Conclusions. — When correleted with the previous data on the metabolic activation of potato tuber discs during incubation (cf also Romberger and Norton the present results suggest that the activation process in potato tuber slices comprehends two distinct phases. In the former, the rise of respiration appears to be a consequence of a release from lack of high energy phosphate acceptors. In the second (somewhat delayed) phase the increased activity of some key respiratory enzymes furtherly enlarges the oxidative capacity of the tissues. The inhibition of this phase of activation together with that of protein synthesis by both DNP or low oxygen tension suggests that enzyme synthesis, rather than enzyme activation, is responsible for the previously reported increase of respiratory enzyme activities.