Action of some ammonium quaternary compounds upon the glucose upkate in yeast

Abstract

In the present work, a study is made of the mode of action of various quaternary ammonium compounds on yeast cells in relation to glucose transport through the membrane and its later consumption inside the cell.

Formulas and germicidal properties of the compounds used are described (table I). Solutions employed in the experiments were previously titrated with a crystallised and purified sodium sulphate lauril, according to Epton’s technique (16) (table II).  

In table III, absolute surface tension values at 19° C. are given for 0.002 M. Solutions of the various compounds. Determinations were brought about by the Lecompte Du Nouy method.

Surface tension diagrams are studied, it being found that di-decyldimethyl-ammonium bromide shows a sharp discontinuity in the slope of the curve for a certain level of concentration (table IV and figure 1). As in other authors (2, 3, 10) it is admitted that the anomaly is a consequence of the micelle formation. It is suggested that apart from the determined critical value, the true concentration is determined by Co being the concentration of free ions, z the number of ions which constitute each micelle and y the fraction.

It is supposed that the formation of micelles from free ions and molecules may take place by the simultaneous intervention of two types of forces: a) mutual attraction between paraffin chains and b) attraction between the water molecules and strongly polar extremes of the tensoactive solute. Stability of the micelles may depend particularly on the covering of hydratation originated by the second type of force.

The degree of hydratation of the micelles may be influenced by the presence of inorganic ions or of certain organic substances with hydroxyl or amine groups. The possibility that the inflection on the surface tension diagram is due to impurities may be set aside.

In tables V, VI and VII and in figure 2, is shown the effect of various inorganic chlorides on the surface tension diagrams of didecyl-dimethylammonium bromide. Results obtained agree with the theoretical forecast and establish the fact that the inflection of the surface tension curve of this quaternary ammonium compound can be suppressed by adding 1 % sodium chloride to the solution.

A stury is made on the effect of the four quaternary ammonium compounds on the absorption of glucose by fresh baker’s yeast (Saccharomyces cerevisiae). As indicated in another publication (39), the procedure followed is fundamentally that of Rothstein and Rarrabee (45). In tables VIII, IX and X and in, figures, 3, 4 and 5, the results obtained are shown (for di-isobutyl - phenoxy-ethoxy-ethyl-dimethyl-benzyl-ammonium chloride, see 39). All show a great inhibitory activity even in very low concentrations. Inhibition grows rapidly and almost linearly.

Inhibition of glucose absorption by di-decyl-dimethyl-ammonium bromide does not increase in proportion to the concentration (figure 9); for certain values of the same, a very pronounced change is shown in the relation between the grade of inhibition and the concentration of inhibitor.

The anomaly presents itself at the same level of concentration as that at which micelle formation takes place (figure I). When the formation of micelles is impeded by sodium chloride, the inhibition curve of didecyldimethyl- ammonium bromide shows a completely normal shape (figure 6). This result allows one to suppose that the anomalous inhibition curve is also a consequence of the formation of micelles.

Influence of cellular concentration on the quantity of glucose taken up from the external medium without inhibitor and in the presence of di-isobutyl, phenoxy-ethoxy-ethyl-dimethyl-benzyl-ammonium chloride has been studied. The data obtained are presented in table XII and in figure 7. Absorption is appreciably proportional to the cellular concentration in the presence or absence of the inhibitor. For a fixed quantity of inhibitor, inhibitions are inversely proportional to the yeast concentrations. It is concluded that these results are only valid when the glucose concentration is almost constant throughout the experiments.

Determinations were brought about of the quantity of di-isobutyl-phenoxy-ethovy-ethyl-dimethyl-benzyl-aninionium chloride taken up by yeast in various initial concentrations of the compound. Results obtained are shown in table XIII and figure 9. A saturation value of the cells for a relatively high value of the quaternary ammonium is found. The quantities of inhibitor which suppress glucose absorption are far below this saturation level.

The theoretical significance of the curve of figure 9 is discussed. Equilibrium between quaternary ammonium cations and the yeast cells is described by the Freundlich isotherm (figure 10). It is concluded that below saturation level, the quantity of inhibitor which penetrates the cell must be very slight compared to that fixed on the cellular membrane. Fixation oi quaternary ammonium compounds according to isotherm adsorption permits one to forecast the consequences of micelle formation.

It is admitted that anomalous inhibition curves have two isotherms: one for the; micellar form, and the other for the ionic or molecular form. Figure 11 permits us to suggest that inhibitor equilibrium between the medium and the membrane is more displaced towards the former in the micellar form than in the ionic form. In figure 12, the theoretical form is represented in the two adsorption isotherms. For a similar increase in concentration in the medium, the concentration effective for the cellular surface increases in very different proportions in each of the two cases.

The influence of sugar concentration on aerobic utilisation of exterior glucose in the presence of di-isobutyl-phenoxy-ethoqy-ethyl-dimethyl-benzyl-ammonium chloride is studied. Results obtained are presented in table XV and in figure 8. A saturation level is found above which, with the quaternary ammonium compound, absorbed glucose not only does not increase but markedly decreases.

It is confirmed that anaerobic absorption of glucose by yeast cells follows the equation of Michaelis-Menten (figure 13). The quaternary ammonium compound acts as a non-competitive inhibitor for the interval of glucose concentrations situated below saturation level.

By previous incubation of yeast cells in a glucose solution, a considerable endogenous alcoholic fermentation can be induced (38, 40). In tables XVI, XVII and XVIII and in figures 3, 4, 5, are shown the results obtained on the effect of quaternary ammonium compounds on endogenous alcoholic fermentation [for di-isobutyl-phenovy-thoxy-ethyl-dimethyl-benzylammonium chloride see (39)]. All are shown to be effective and inhibition grows very rapidly when the product begins to act, following an approximately linear, course.

It is admitted that a certain substance acts on the passage across the membrane when it is capable of inhibiting absorption without affecting endogenous alcoholic fermentation (39). In table XIX, are found values of selectivity on membrane obtained for the four quaternary ammonium compounds. As in the theoretical forecast, all show a high index of inhibition of glucose transfer across the cellular membrane. In table XIX and in figure 11, is shown a different selectivity on membrane for free ions and for the micelles, of didecyl-dimethyl-ammonium bromide.

Capillary activity of the four compounds studied (table III), shows no relation to their respective membrane selectivity (table XIX). The Systems which control the passage of glucose through the membrane in yeast must be unequally influenced by each of the substances studied.

Surface activity of the four quaternary ammonium compounds has no relation to the absolute activity on utilisation of exterior glucose, nor to the inhibition of endogenous fermentation (table III and table XX).

Parallel with their germicidal power (42) and their inhibition of respiration and glucose fermentation by various microorganisms (6), compounds which possens the benzyl group in their molecule are those which show most selectivity on membrane.

As had been found for germicidal power (42), the two compounds most active on membrane have practically the same length of side chain. It is very probable that there exists a critical length oi the lipophil group linked to the phenomenon of micelle formation.

Results obtained permit one to suggest that the curve oi absorption inhibition must be equivalent to the curve of inhibition of endogenous fermentation plus the membrane factor Both are unequally affected on varying the concentration oi inhibitor in the medium.

Three quaternary ammonium compounds are shown to be equally toxic for the enzymatic systems included in endogenous alcoholic fermentation. Cetyl-trimethyl-ammonium bromide is lees active.

With didecyl-dimethyl-ammonium bromide al concentrations which begin to act in the interior of the cell a change of slope exists which is markedly accentuated by the curve of absorption inhibition. Complete suppression of exterior glucose utilisation is only reached for concentrations more than twice as great as that necessary to totally inhibit endogenous metabolism (figure 5). It is admitted that this is a consequence on the one hand of the fact that glucose penetration in the cell is acted upon by the micellar form, while on the other hand endogenous processes are acted upon by the ionic form. However, penetration of glucose is less sensitive to the micelles than the glucogenolysis' to the ions.

The four quaternary ammonium compounds studied act primarily on the cellular membrane inhibiting the passage of glucose. Above a certain concentration, they later inhibit the endocellular systems of anaerobic glucidic metabolism. As a matter of fact, they behave as true enzymatic inhibitors capable of penetrating the cell and endowed with little specificity. Selective action on glucose transfer through the membrane must be a consequence of the high-concentration of inhibitor at the level of cell surface.