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Kinetical parameters of monovalent cation uptake in yeast are calculated according to a model for mutual interaction of membrane potential and cation uptake. Apparent Km values for monovalent cation uptake obtained from uptake studies are 3-5-times lower than the Km values expected when the membrane potential remains constant at increasing cation concentrations instead of being reduced. The model accounts for various phenomena as (i) the increase in apparent Km of Rb+ uptake accompanying the decrease in maximum rate of uptake found on increasing the cellular K+ content, (ii) the decrease in maximum uptake rate and increase in Km in case of simultaneous transport of phosphate and Rb+, (iii) the change in the maximum uptake rate without a change in Km under conditions that the proton pump is effected and (iv) the absence of an increase in K+ efflux at high depolarizing external cation concentrations.

Inositol 1,4,5-trisphosphate (IP3) induces a release of Ca2+ from vacuolar membrane vesicles of Saccharomyces cerevisiae. The amount released is dependent on IP3 concentration (concentration for half maximal effect, Km, apparent = 0.4 microM). Myo-inositol, and inositol 1,4-bisphosphate up to 50 microM have no effect on Ca2+ levels in the vesicles. The IP3-induced Ca2+ release is blocked by dantrolene and 8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate-HCl (TMB-8), which are known to block Ca2+ release from Ca2+ stores in animal cells. IP3-induced release of Ca2+ also occurs when Ca2+ is accumulated by means of an artificial pH gradient, indicating that the effect of IP3 is not due to an effect on the vacuolar H(+)-ATPase. The IP3-induced Ca2+ release is not accompanied by a change in the pH gradient, which indicates that it is not due to a reversal of the Ca2+/nH+ antiport or to a decrease in delta pH by IP3. The present results suggest that IP3 may act as a second messenger in the mobilization of Ca2+ in yeast cells. As in plant cells, the vacuolar membrane of yeast seems to contain a Ca2+ channel, which can be opened by IP3. In this respect the vacuole could function as an IP3-regulated intracellular Ca2+ store, equivalent to the endoplasmic- and sarcoplasmic reticulum in animal cells, and play a role in Ca(2+)-dependent signal transduction in yeast cells.

The concentration dependence of cation uptake by the cell may be considerably complicated when this uptake is accompanied by a depolarization of the cell membrane. In case of carrier-mediated transport deviations from Michaelis-Menten kinetics may come to the fore comparable to those found in a dual mechanism of cation uptake or when substrate inhibition is involved. This remains true when only the maximum rate of uptake and not the Km is dependent upon the membrane potential. We have proven this by means of computer simulation of cation transport mediated by a non-mobile carrier. Under restricted conditions still apparent Michaelis-Menten kinetics may be found despite the fact that the membrane potential varies with increasing substrate cation concentration. But even then there are still differences with 'normal' transport kinetics. A non-competitive inhibitor does not only affect the maximum rate of uptake but also the apparent Km. Depolarization of the cells by a cation which passes the cell membrane by means of diffusion, affects the uptake of the substrate cation almost in the same way as a non-competitive inhibitor does and causes both a decrease in the maximum rate of uptake and an increase in Km. In the case of competitive inhibition the apparent affinity of the inhibitor for the carrier depends upon the rate of transfer of this inhibitor through the cell membrane. The mutual influence of cation uptake and membrane potential is dealt with for uniport of either monovalent or divalent cations and for cotransport of monovalent cation with protons, as well. Possible effects of the surface potential are accounted for.

The transient increase in the rate of 2-(4-di-methyl-aminostyryl)-1-ethylpyridinium (DMP) influx in Saccharomyces cerevisiae caused by addition of glucose to a suspension of non-metabolizing cells at relatively high pH can be prevented by monovalent cations. Their concentrations for half-maximum inhibition of DMP uptake are of the same order of magnitude as the corresponding Km values for their uptake into yeast cells. It is argued that the inhibition of DMP uptake by monovalent cations is caused by a fast depolarization of the cell membrane and a second further decrease in the rate of DMP uptake. The latter effect develops slowly with time and depends upon the extent of accumulation of the monovalent cations in the cells.

Monovalent cations decrease the initial rate of uptake of the membrane potential probe 2-(dimethylaminostyryl)-1-ethyl-pyridinium (DMP) into metabolizing cells, showing that the cells are depolarized. A steep decrease in this rate was found even at low cation concentrations, reaching 62%, 42%, 58%, 40% and 40% at high concentrations of K+, Rb+, Cs+, Na+ and Li+, respectively. The corresponding concentrations at which half-maximum decrease was found were 0.22, 0.36, 1.2, 17 and 17 mM. These values are of the same order of magnitude as the half-saturation concentrations for monovalent cation uptake by the yeast.

At pH 7, addition of glucose to an anaerobic suspension of non-metabolizing yeast cells causes a transient net efflux of K+ from the cells and a concomitant transient hyperpolarization of the plasma membrane (Van de Mortel, J.B.J., et al. (1988) Biochem. Biophys. Acta 936, 421-428). Both phenomena are effectively suppressed in the presence of low concentrations of polyvalent cations. The concentrations of Mn2+, Ca2+, Ba2+, Mg2+, Sr2+ and La3+ required for half-maximal suppression of the transient hyperpolarization are 10, 17, 20, 38, 47 and 5 microM, respectively. Subsequent addition of EDTA 90 s after that of Ca2+ immediately restores both K+ efflux and cellular uptake of the fluorescent membrane potential probe 2-(dimethylaminostyryl)-1-ethylpyridinium (DMP). This suggests that an interaction of polyvalent cations with an external binding site blocks the putative K(+)-selective channel. Opening of this channel is not blocked by 20 mM tetraethylammonium nor by 100 microM 3,4-diaminopyridine. It is argued that this glucose-induced K(+)-conductive pathway is not identical to the voltage-gated K+ channels identified until now in patch-clamp studies of the yeast plasma membrane.

(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5.

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.

Phosphate starvation derepresses a high-affinity phosphate uptake system in Saccharomyces cerevisiae strain A294, while in the same time the low-affinity phosphate uptake system disappears. The protein synthesis inhibitor cycloheximide prevents the derepression, but has no effect as soon as the high-affinity system is fully derepressed. Two other protein synthesis inhibitors, lomofungin and 8-hydroxyquinoline, were found to interfere also with the low-affinity system and with Rb+ uptake. After incubation of the yeast cells in the presence of phosphate the high-affinity system is not derepressed, but the Vmax of the low-affinity system has decreased from about 35%. Phosphate supplement after derepression causes the high-affinity system to disappear to a certain extent while in the meantime the low-affinity system reappears. The results are compared with those found in the yeast Candida tropicalis for phosphate uptake.

Membrane potentials of yeast cells, Saccharomyces cerevisiae, calculated from the equilibrium distribution of tetraphenylphosphonium (TPP) between cell-water and medium should be corrected for a contribution due to binding of TPP to intracellular constituents. The magnitude of this correction depends upon the way in which it is determined. In cells permeabilized by boiling, cell-binding is much higher than in cells permeabilized by repeated freezing and thawing. The binding corrections are 75 +/- 1 mV and 49 +/- 7 mV, respectively. The binding correction obtained from TPP distribution between deenergized cells and medium is much lower and amounts to 19 +/- 9 mV. The latter value is probably more reliable. It is supposed that permeabilization of the cells by boiling or repeated freezing and thawing unmasks potential TPP binding groups in the cell. The K+ accumulation into anaerobically metabolizing yeast cells can be accounted for almost quantitatively by a cotransport of protons and K+ ions if the lower binding correction is applied. This means that K+ accumulation into the yeast cell may be driven by the sum of the protonmotive force and the membrane potential.

The distribution ratio of the lipophilic cation dibenzyldimethylammonium between the cells of Saccharomyces cerevisiae and the medium appears to reflect changes in the membrane potential in a way that is qualitatively correct: the addition of a proton conductor or of an agent which blocks metabolism causes an apparent depolarization of the cell membrane; monovalent cations cause also a lowering of the equilibrium distribution, whereas the addition of divalent cations results in an increase of the partition ratio. However, uptake of dibenzyldimethylammonium and probably also of other liophilic cations proceeds via the thiamine transport system of the yeast. Dibenzyldimethylammonium transport is inducible, like thiamine transport. A kinetic analysis of the mutual interaction between thiamine and dibenzyldimethylammonium uptake shows that these compounds share a common transport system; moreover, dibenzyldimethylammonium uptake is inhibited complete by thiamine disulfide, a competitive inhibitor of thiamine transport and dibenzyldimethylammonium uptake in a thiamine-transport mutant is reduced considerably. It is concluded that one should be cautious when using lipophilic cations to measure the membrane potential of cells of S. cerevisiae.

The concentration dependence of the uptake rate of divalent cations in yeast can be described by a simple diffusion process after accounting for the effect of the surface potential upon the divalent cation concentration near the membrane. It is also necessary to correct for the effect of the cell pH upon the rate of translocation. The apparent saturation kinetics is ascribed to the fact that the quotient of the concentration of the divalent cations near the cell membrane and the bulk aqueous phase concentration is reduced on increasing the divalent cation concentration in the medium. The diffusion process regulated by the surface potential even mimics the saturation kinetics of a two-carrier transport system. The selectivity found between Ca2+ and Sr2+ uptake can probably be traced to differences in their affinity for the negative groups on the cell membrane determining the surface potential rather than to differences in their affinity for a transport system. The enhancement of divalent cation uptake by loading the cells with phosphate is probably due to the concomitant increase in the net negative charge of the cell membrane.

The uptake of monovalent cations by yeast via the monovalent cation uptake mechanism is inhibited by phosphate. The inhibition of Rb+ uptake shows saturation kinetics and the phosphate concentration at which half-maximal inhibition is observed is equal to the Km of phosphate for the sodium-independent phosphate uptake mechanism. The kinetic coefficients of Rb+ and TI+ uptake are affected by phosphate: the maximal rate of uptake is decreased and the apparent affinity constants for the translocation sites are increased. In the case of Na+ uptake, the inhibition by phosphate may be partly or completely compensated by stimulation of Na+ uptake via a sodium-phosphate cotransport mechanism. Phosphate effects a transient stimulation of the efflux of the lipophilic cation dibenzyldimethylammonium from preloaded yeast cells and a transient inhibition of dibenzyldimethylammonium uptake. Possibly, the inhibition of monovalent cation uptake in yeast can be explained by a transient depolarization of the cell membrane by phosphate.

The uptake of Ca2+ and Sr2+ by the yeast Saccharomyces cerevisiae is energy dependent, and shows a deviation from simple Michaelis-Menten kinetics. A model is discussed that takes into account the effect of the surface potential and the membrane potential on uptake kinetics. The rate of Ca2+ and Sr2+ uptake is influenced by the cell pH and by the medium pH. The inhibition of uptake at low concentration of Ca2+ and Sr2+ at low pH may be explained by a decrease of the surface potential. The inhibition of Ca2+ and Sr2+ uptake by monovalent cations is independent of the divalent cation concentration. The inhibition shows saturation kinetics, and the concentration of monovalent cation at which half-maximal inhibition is observed, is equal to the affinity constant of this ion for the monovalent cation transport system. The inhibition of divalent cation uptake by monovalent cations appears to be related to depolarization of the cell membrane. Phosphate exerts a dual effect on uptake of divalent cations: and initial inhibition and a secondary stimulation. The inhibition shows saturation kinetics, and the inhibition constant is equal to the affinity constant of phosphate for its transport mechanism. The secondary stimulation can only partly be explained by a decrease of the cell pH, suggesting interaction of intracellular phosphate, or a phosphorylated compound, with the translocation mechanism.

The effect of bivalent cations on phosphate uptake by Saccharomyces cerevisiae was investigated. Phosphate uptake via the Na+-dependent transport system at pH 7.2 is stimulated by bivalent cations. The apparent affinity of phosphate for the transport mechanism is increased, but the apparent affinity for Na+ is decreased. Uptake of phosphate via the Na+-independent transport system is accompanied by a net proton influx of 2H+ and an efflux of 1 K+ for each phosphate ion taken up. At pH 4.5 phosphate uptake via the Na+-independent system is stimulated by bivalent cations, whereas at pH 7.2 uptake is inhibited. The effect of bivalent cations on phosphate uptake can be ascribed to a decrease in the surface potential.

The larger accumulation of Mn2+ than of Sr2+ in Saccharomyces cerevisiae is ascribed to the operation of a specific extrusion pump, presumably a Ca2+ pump, which has a higher affinity for Sr2+ than for Mn2+. The differences in accumulation levels of Mn2+ and Sr2+ attained after prolonged incubation are completely abolished in cells of which the plasmamembrane has been permeabilized with the polybase DEAE-dextran under isotonic conditions. In the permeabilized cells Sr2+ and Mn2+ accumulation levels are attained as for Mn2+ in intact cells. It is suggested that the accumulation of divalent cations into the permeabilized cells mainly represents their accumulation into the vacuoles. Also the cationic dye ethidium abolishes the differences in Mn2+ and Sr2+ accumulation. The dye increases the accumulation of Sr2+ but decreases that of Mn2+ somewhat. It cannot be distinguished yet whether its action is due to an impairment of the efflux pump or to an increase in the permeability of the plasmamembrane facilitating the divalent cations to be accumulated into the vacuoles. Ethidium does not affect the initial rates of divalent cation uptake into the vacuoles, but it effectively reduces the ultimate accumulation of the divalent cations in the DEAE-dextran permeabilized cells, possibly by competing with the divalent cations for intravacuolar binding sites. Similar results are obtained for the accumulation of Ca2+. It is concluded that the efflux pump enables the yeast cell to regulate accumulation levels of the various divalent cations to different extents.

The enhancement of divalent cation uptake in yeast provoked by the membrane ATPase inhibitors trifluoperazine, miconazole, compound 48/80, ethidium, DIO-9 and calmidazolium should be ascribed to an increase in cation permeability of the yeast rather than to hyperpolarisation of the yeast cell membrane. For trifluoperazine and miconazole it is unequivocally shown that the cells are hyperpolarized though for miconazole only transiently. Whether the other drugs also hyperpolarize the yeast cells is uncertain. The apparent hyperpolarisation caused by trifluoperazine and miconazole may be attributed to a specific increase in the K+ permeability of the yeast plasmamembrane evoked by these compounds.

The characteristics of the uptake of the lipophilic cation tetraphenylphosphonium (TPP+) into Saccharomyces cerevisiae have been investigated in order to establish whether this compound can be used to monitor the membrane potential of his organism. Unlike dibenzyldimethylammonium, TPP+ is not translocated via the thiamine transport system, nor via another inducible translocation mechanism. On changing the experimental conditions the equilibrium potential of TPP+ varies according to expected changes of the membrane potential. TPP+ accumulation is higher in metabolizing cells than in non-metabolizing cells. In addition, decreasing the medium pH, addition of the proton conductor 2,4-dinitrophenol and addition of K+ all cause an apparent depolarization, whereas Ca2+ apparently hyperpolarizes the cell membrane. It is concluded that TPP+, if applied at low concentrations, can be used to measure the membrane potential of S. cerevisiae.

Phosphate uptake by yeast at pH 7.2 is mediated by two mechanisms, one of which has a Km of 30 micronM and is independent of sodium, and a sodium-dependent mechanism with a Km of 0.6 micronM, both Km values with respect to monovalent phosphate. The sodium-dependent mechanism has two sites with affinity for Na+, with affinity constants of 0.04 and 29 mM. Also lithium enhances phosphate uptake; the affinity constants for lithium are 0.3 and 36 mM. Other alkali ions do not stimulate phosphate uptake at pH 7.2. Ribidium has no effect on the stimulation of phosphate uptake by sodium. Phosphate and arsenate enhance sodium uptake at pH 7.2. The Km of this stimulation with regard to monovalent orthophosphate is about equal to that of the sodium-dependent phosphate uptake. The properties of the cation binding sites of the phosphate uptake mechanism and those of the phosphate-dependent cation transport mechanism have been compared. The existence of a separate sodium-phosphate cotransport system is proposed.

Uptake of sulfate by yeast requires the presence of a metabolic substrate and is dependent on the time during which the cells have been metabolizing in the absence of sulfate. At low concentrations of sulfate, uptake can be described by simple saturation kinetics. Uptake of sulfate is accompanied by a net proton influx of 3 H+ and an efflux of 1 K+ for each sulfate ion taken up. Divalent cations stimulate sulfate uptake at low concentrations of sulfate; the maximal rate of uptake is not significantly affected but Km is lowered. Stimulation by divalent cations shows an optimum at a cation concentration of about 4 mM. Monovalent cations are less effective, trivalent cations are more effective in stimulating sulfate uptake. The results are qualitatively in accordance with the notion, that the effect of cations is due to an effect via the surface potential.

The initial rate of both Ca2+ and Mn2+ uptake is inhibited by ruthenium red to about the same extent as by equivalent concentrations of La3+. The inhibition of Ca2+ uptake, however, is relieved during further incubation with ruthenium red. On preincubating the cells with ruthenium red even a stimulation of divalent cation uptake can be found. Relieve of the inhibition of divalent cation uptake is accompanied by K+ efflux. Both ruthenium red and La3+ displace Ca2+ very effectively from binding sites at the cell surface. The inhibition of initial Ca2+ uptake is accompanied by a reduction in the binding of Ca2+.

The effect of Cd2+ poisoning of Saccharomyces cerevisiae on 45Ca, 109Cd and [14C]tetraphenylphosphonium (TPP) uptake and cell pH was examined. At Cd2+ concentrations that produced substantial K+ efflux the rates of uptake of 45Ca, 109Cd and [14C]TPP increased progressively during incubation of the cells with Cd2+, and the cell pH was lowered concomitantly. The initial rates of uptake of the divalent cations and of TPP were increased in cells pre-loaded with Cd2+, which shows that stimulation of the ion fluxes was exerted by the Cd2+ that accumulated in the cells. The distribution ratio of TPP between cells and medium, however, was decreased by Cd2+. Although hyperpolarization of the cell membrane by Cd2+ cannot be excluded, it is argued that Cd2+ primarily stimulated divalent cation uptake by increasing the cation permeability of the cell membrane allowing the cations to enter the cells more easily.

1. Both the maximum rate of phosphate uptake and the Km depend upon the pH of the medium in a complex way. 2. The effect of medium pH upon the maximum rate of uptake is mainly indirect and is correlated with changes in cell pH. 3. The Km is affected by the medium pH both directly via an apparent competitive inhibition by hydroxyl anions and indirectly in a similar way as the maximum rate of uptake.

At pH 7, addition of glucose under anaerobic conditions to a suspension of the yeast Saccharomyces cerevisiae causes both a transient hyperpolarization and a transient net efflux of K+ from the cells. Hyperpolarization shows a peak at about 3 min and a net K+ efflux at 4-5 min. An additional transient hyperpolarization and net K+ efflux are found after 60-80 and 100 min, respectively. Addition of 2-deoxyglucose instead of glucose does not lead to hyperpolarization of the cells or K+ efflux. At low pH, neither transient hyperpolarization nor a transient K+ efflux are found. With ethanol as substrate and applying aerobic conditions, both a transient hyperpolarization and a transient K+ efflux are found at pH 7. The fluorescent probe 2-(dimethylaminostyryl)-1-ethylpyridinium appears to be useful for probing changes in the membrane potential of S. cerevisiae. It is hypothesized that the hyperpolarization of the cells is due to opening of K+ channels in the plasma membrane. Accordingly, the hyperpolarization of the cells at pH 7 is almost completely abolished by 1.25 mM K+, whereas the same amount of Na+ does not reduce the hyperpolarization.

The concentration dependence of both Rb+ uptake and Na+ uptake by yeast can be described by a quadratic rate equation. This equation is derived for translocation of cations via a two-site translocation system. In accordance with predictions made for such a two-site translocation system the shape of the uptake isotherm depends both upon the substrate cation species and upon the concentration of other added competing cations. On plotting the rate of Rb+ uptake against the quotient of that rate and the Rb+ concentration concave, convex and also linear curves are found depending upon the type and the concentration of added monovalent cations. The Na+ uptake isotherm plotted in a similar way shows a shift from a concave curve to a straight line on adding increasing amounts of Rb+ to the yeast suspension. Decreasing the pH of the medium leads to a more pronounced convex uptake isotherm for Rb+ uptake. This can be ascribed to an indirect effect of the surface potential upon Rb+ uptake and to occupation of the binding sites by protons, which are replaced again by Rb+ at increasing Rb+ concentrations. Replacement of one sodium ion from one of the two sites of the monovalent cation transport system by either K+ OR Rb+ leads to an increase in the rate of Na+ translocation across the yeast cell membrane.

The inhibitors of the yeast plasma membrane ATPase, Dio-9, miconazole, suloctidil, N,N'-dicyclohexycarbodiimide, triphenyltin and diethylstilbestrol provoke an all-or-none K+ loss from the cells. Some of the K+-depleted cells also show an increased permeability for the dye, Bromophenol blue, indicating that the barrier properties of these cells are drastically changed. Apart from this all-or-none process, a graded loss of K+ is also observed. These observations warn against the use of the inhibitors in studies aimed at evaluating the role of the ATPase in the energy transduction of membrane transport processes of yeasts.

In experiments dealing with the effect of xenobiotics upon the efflux of K+ from yeast cells, one should be aware that when this efflux proceeds via an all-or-none process, the K+ being released from the intoxicated cells can again be accumulated into the still unaffected cells. Therefore, the measured net efflux of K+ will be less than the efflux from the intoxicated cells. The difference between these two magnitudes can be minimalized by incubating the cells for only a short period and on applying yeast densities that are not too high. When the cells are permeabilized relatively slowly but ultimately to a great extent, the kinetics of K+ efflux may be quite complicated.

The rate of uptake of the lipophilic cation dibenzyldimethylammonium by yeast cells is increased by tetraphenylboron. However, tetraphenylboron increases also the equilibrium partition of dibenzyldimethylammonium between cells and medium, probably because a complex between tetraphenylboron and dibenzyldimethylammonium is trapped inside the cells. Accumulation of dibenzyldimethylammonium in the presence of tetraphenylboron is not reversed by dinitrophenol, whereas accumulation of the lipophilic cation in the absence of tetraphenylboron appears to be almost completely reversible.

Stimulation of K+ efflux from non-metabolizing yeast cells by 2,4-dinitrophenol or by salicylic acid occurs only after accumulation of the compounds into the cells, indicating that the site of action of the uncouplers is inside the cells. A correlation is found between the partition ratio of the lipophilic cation dibenzyldimethylammonium between cells and medium and the rate of K+ efflux.

The effect of changes in the intracellular pH upon the concentration dependence of the Rb+ uptake by yeast is investigated. It is shown, that the uptake of Rb+ can be described by a mechanism in which the total concentration of primary binding sites at the outer side of the membrane is independent of the intracellular ligand composition and of the membrane potential, and the influx rate constants depend upon the intracellular pH and/or upon the membrane potential. It is argued that the involvement of a mobile carrier mechanism is not likely.

Initial uptake of Mn2+ and Sr2+ in the yeast Saccharomyces cerevisiae was studied in order to investigate the selectivity of the divalent cation uptake system and the possible involvement of the plasma-membrane ATPase in this uptake. The initial uptake rates of the two ions were not significantly different. This ruled out a direct role of the plasma-membrane ATPase, since this ATPase is specific for Mn2+ compared to Sr2+. After 1 h uptake, Mn2+ had accumulated 10-times more than Sr2+. Influx of Mn2+ and Sr2+ remained unchanged during that time, however. The differences in accumulation level found for Mn2+ and Sr2+ could be ascribed to a greater efflux of Sr2+ as compared with Mn2+. Probably this greater efflux of Sr2+ was only apparent, since differential extraction of the yeast cells revealed that Mn2+ is more compartmentalised than Sr2+, giving rise to a lower relative cytoplasmic Mn2+ concentration.

Addition of a macromolecule to a solution will give rise to a large excluded volume for the centers of the solute molecules. This will cause an apparent increase in solute concentration which is of the same order of magnitude as that associated with the nonsolvent volumes reported in the literature. A critical examination of one of the procedures used for the determination of nonsolvent water-the vapor pressure method of Hill-is given, and it is concluded that, with the use of this method, it is impossible to detect any significant nonsolvent water surrounding bovine albumin for either sugars or polyols. Generally, data reported in the literature for the nonsolvent water of proteins or other macromolecules will be too high unless they are corrected for the excluded volume.