Separation-out of the effect upon membrane in sugar uptake inhibition in yeast

Abstract

Ordinarily, it is difficult to discern whether an inhibitor of glucose uptake acts exclusively or preponderantly on penetration through the membrane, os exercises its action in the cell-interior. Here we discus the recourses utilised by various authors (2, 4, 9), to show that the uranyl ion inhibits the utilisation of exterior glucose by acting exclusively on penetration through the cell-membrane.

After having succeeded in inducing spontaneous alcoholic fermentation in yeast (5, 7), in the present work the author suggests the possibility of isolating functionally in a general way, the effect of an inhibitor on the passage of glucose through the membrane from all other action which could be brought about simultaneously inside the cell.

Firstly the action of the problem substance on utilisation of external glucose is studied (5). The following is a scheme of the operations used:

1. Previous treatment to evacuate reserves.


2. Purification of yeast. 3. Adjustment of the pH to between 3 and 4.


3. Determination of sediment and adjustment of cell concentration to a final value of 0.03 mi./mi. (14.253 mg./ml.).


4. Thirty minutes of anaerobic absorption at 30°C. and with, an initial glucose concentration of 0.3%.


5. Centrifugation.


6. Determination of glucose in the supernatant liquid.


7. This test permits one to determine the glucose absorbed without inhibitor and in the presence of a certain quantity of inhibitor. The percentage of inhibition can be calculated from these two values. In table I and figure 1 (A) the results obtained for diisobutyl-phenoxy-ethoxy-ethyl-dime thyl-benzylammonium chloride are given.

When the action of the inhibitor on the utilisation of the exterior glucose is known, the studv of its action on alcoholic endogenous fermentation is proceeded with. Por this the following technique is followed:

1. 5 g. of compressed yeast are weighed and are dispersed as perfectly as possible in distilled water. In another container 5 g. of glucose are dissolved in distilled water. The two portions are mixed and the volume is brought to 100 ml. with distilled water.


2. The yeast suspension is kept a bath at 30°C. for 1 hour. During all this time, it is stirred and the partial pressure of oxygen is kept contant by continuous injection of a strong current of air.


3. The incubated yeast is centrifuged. The sediment obtained is washed twice with distilled water.


4. The pH is adjusted to between 3 and 4.


5. The sediment is determined and cellular concentration is fixed for a final value of 0.03 mi./mi. (14.253 mg./ml.).


6. 4 ml. of suspension are set in Warburg jars.


7. The air is displaced by the passage of purified nitrogen for 1/2 hour.


8. The microlitres of carbon dioxide given off in a half-hour at 30°C. are measured.

In table II and fig. 1 (B) are given the results obtained for di-isobutyl- phenoxy-ethoxy-ethyl-dimethyl-benzyl-ammonium chloride.

Uranyl nitrate (5), sodium fluoride (5), and di-isobutyl-phenoxy-ethoxyethyl-dimethyl-benzyl-ammonium chloride are examples of the three possibilities which exist with respect to the action of an inhibitor on membrane : exclusive inhibition (uranyl), selective inhibition (quaternary amnionium compound) and no inhibition (fluoride).

The action of florricine on the utilisation of exterior glucose by yeast is not found until conccntrations of 1/500 M, are reached (table IV).

So that the scheme of figure 2 may be fundamentally valid, it is necessary that the experiments be performed in an acid medium (pH 3.9). On the other hand, in aerobic conditions or in the presence of others effectors, actions on the endogenous process and on the utilisation of exterior glucose would be difficult to compare.