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G F Fuhrmann

Publications and source records attributed to G F Fuhrmann.

At least 19 recordsLinked to original sources
PubMed1997

Different activation energies in glucose uptake in Saccharomyces cerevisiae DFY1 suggest two transport systems.

The analysis of initial glucose uptake in Saccharomyces cerevisiae at 25 degrees, 20 degrees, 15 degrees and 10 degrees C by computer-assisted nonlinear regression analysis predicts two transport systems. The first demonstrates Michaelis-Menten kinetics and the second shows first order behaviour. The activation energies of these two systems were calculated by the Arrhenius equation at four different growth phases, namely early exponential (EE), middle exponential (ME2), late exponential (LE) and early stationary (ES) with 2% glucose in the batch medium. The activation energies calculated from the V(m) values in EE, ME, LE and ES growth phases were 15.8 +/- 1.7, 13.5 +/- 1.0, 15.1 +/- 0.8 and 13.5 +/- 0.7 kcal/mol. These values are in agreement with activation energies calculated for the first mechanism, facilitated diffusion, which is the mechanism deduced from countertransport experiments. The activation energies derived for the second transport system from the first order rate constants in cells grown to EE, ME2, LE and ES were 8.0 +/- 2.1, 8.1 +/- 1.3, 9.6 +/- 3.0 and 7.5 +/- 2.6 kcal/mol. These values are still significantly higher than for free diffusion of glucose in water and lower as predicted for passage of glucose through the lipid phase. Therefore, we assume in addition to carrier-mediated facilitated diffusion the entrance of glucose into the cell through a pore.

Biological Transport↗
PubMed1995

Iron sequestration by the yeast vacuole. A study with vacuolar mutants of Saccharomyces cerevisiae.

To investigate iron uptake by the yeast vacuole, we examined iron accumulation and subcellular distribution in two Saccharomyces cerevisiae mutant strains with distinct alterations in vacuolar morpholgy and functions, the vacuolar protein targeting mutants vpt13 and vpt16. The mutant vpt13 is deficient in vacuolar acidification but possesses a morphologically normal vacuole, whereas vpt16 is devoid of any vacuole-like structure. The vacuole-lacking mutant vpt16 displayed reduced iron accumulation at high and low extracellular iron concentrations. Iron accumulation by vpt13 was not reduced at low extracellular iron concentrations, and only slightly reduced at high external iron. Subcellular fractionation of vpt13 after iron loading confirmed the vacuolar localization of the accumulated iron. Vacuolar iron sequestration thus is not dependent on vacuolar acidification, in contrast to vacuolar uptake of other metal cations such as calcium, strontium, manganese, and zinc. Conclusively, the vacuolar iron-uptake mechanism is different from the cation-proton exchangers mediating uptake of these other metal cations. Regarding the lacking dependence on vacuolar acidification as well as the high amounts of iron accumulated by the vacuole, vacuolar iron uptake could be mediated by an iron pump similar to the known ATP-dependent mechanisms of active transport.

Adenosine Triphosphate↗
PubMed1993

Misuse of graphical analysis in nonlinear sugar transport kinetics by Eadie-Hofstee plots.

It has become common practice to analyse the sugar transport kinetics from initial uptake rates in Saccharomyces cerevisiae cells with Eadie-Hofstee plots. These plots often demonstrate a nonlinear behaviour. They have been resolved incorrectly into two quasilinear components indicating the presence of (at least) two uptake systems or components, with Km values differing by a factor of about 10. This graphical analysis neglects the obvious additivity of the two hypothetical systems and is therefore in error. A more efficient way to determine kinetic parameters from initial uptake experiments is to use computer-assisted nonlinear regression analysis.

Glucose↗
PubMed1992

Phloretin keto-enol tautomerism and inhibition of glucose transport in human erythrocytes (including effects of phloretin on anion transport).

Under various pH conditions phloretin demonstrates keto-enol tautomerism with a pK value of 7.26 +/- 0.06. As Wilbrandt has shown ((1950) Arch. Exp. Pathol. Pharmacol. 212, 9-29) phloretin added to erythrocytes inhibits glucose efflux, but not glucose influx. At pH 6.5 a Ki value of 0.36 and at pH 9 of 22.7 microM was measured; only the ketonic form of phloretin contributes to the inhibition of glucose efflux. This was also the case for inhibition of galactose efflux and anion exchange. The geometry optimization of a large number of conformations of the ketonic and enolic forms of phloretin demonstrates different shapes of the molecules. Only the ketonic form shows several overlapping structures with beta-D-glucopyranose. Considering surplus binding of phloretin under glucose efflux conditions as being equivalent to the number of glucose transporters, a number of about 200,000 molecules was determined. By two independent methods 210,000 and 171,000 molecules per cell were calculated. This result is in close agreement with the number of glucose transporter sites of the erythrocyte.

Bicarbonates↗
PubMed1990

Vanadium uptake by yeast cells.

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.

Cations, Divalent↗
PubMed1989

Kinetic analysis and simulation of glucose transport in plasma membrane vesicles of glucose-repressed and derepressed Saccharomyces cerevisiae cells.

In this study experimental data on the kinetic parameters investigated by other authors 1-5, 11 together with own data on plasma membrane vesicles, have been subjected to a computer simulation based on the equations describing facilitated diffusion. The simulation led to an ideal fit describing the above data. From this it can be concluded that glucose is transported by facilitated diffusion, and not by active transport as was postulated by Van Steveninck 14,15. The simulation method also demonstrates that the fast sampling technique used by these authors 1-5, 11 underestimated the fluxes. Thus, the parameters given do not contribute to the understand of glucose transport under different metabolic conditions. The K value of plasma membrane vesicles prepared from glucose-repressed cells is around 7 mM. Derepression, particularly by galactose, causes a highly significant increase in affinity as shown by a decrease in the K value to 2 mM. The highest affinity was measured in a triple kinaseless mutant grown on glycerol with a K value of 1 mM. It seems, therefore, that the kinetic parameters derived from initial uptake rates of glucose in intact cells 1-5, 11 using single flux analysis, such as Eadie-Hofstee- or Lineweaver-Burk-plots, are in error.

Biological Transport↗
PubMed1989

The effect of ferricyanide with iodoacetate in calcium-free solution on passive cation permeability in human red blood cells: comparison with the Gardos-effect and with the influence of PCMBS on passive cation permeability.

Freshly prepared human red blood cells incubated with 5 mM ferricyanide, 0.2 mM iodoacetate and 2 mM adenosine in the presence of 5 mM EGTA demonstrate comparable increases in Na+ and K+ permeability (ferricyanide effect). This effect is unrelated to the Ca2+-activated K+ channel (Gardos effect) since influx of Ca2+ from outside the cell is excluded. Also this effect is different from the non-specific Na+ and K+ permeability change elicited by PCMBS. These differences become obvious by using various reagents. For example, A23187 and quinidine exert opposite effects in Gardos and ferricyanide experiments, where A23187 and atebrin react oppositely in the latter and in PCMBS experiments. The ferricyanide effect described here does not involve formation of nonspecific channels. The change in Na+ permeability separately from K+ permeability under certain circumstances suggests a more specific effect.

4-Chloromercuribenzenesulfonate↗
PubMed1989

Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. I. Effects of propranolol.

To study the effect of propranolol on the Ca2+- or Pb2+-activated K+ permeability in human erythrocytes, K+ effluxes were compared with single-channel currents. The results demonstrate that propranolol has a twofold effect: (1) it renders the channel protein more sensitive to Ca2+ or Pb2+; and (2) it simultaneously inhibits channel activity and slightly reduces single-channel conductance. The number of active channels is not affected.

Calcimycin↗
PubMed1989

Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. II. Parallelisms to modulation of the activity of a membrane-bound oxidoreductase.

Modulation of Ca2+-activable K+ permeability was compared with modulation of a membrane-bound oxidoreductase activity in human erythrocytes. Changes in the K+ permeability were monitored by flux measurements and single-channel recordings. The enzyme activity was detected by measuring reduction of ferricyanide. Pb2+, Atebrin and menadione had parallel effects on the channel protein and the enzyme. In contrast, propranolol stimulates K+ permeability, but is without effect on enzyme activity. The results demonstrate that the K+ channel and the enzyme are distinct membrane proteins but that the enzyme activity may influence channel gating.

Calcium↗
PubMed1988

The anion-transport inhibitor H2DIDS cross-links hemoglobin interdimerically and enhances oxygen unloading.

Human hemoglobin treated with equal concentrations of the anion-transport inhibitor H2DIDS produces a right shift in the oxygen dissociation curve. concomitantly, the Hill coefficient is reduced from n = 2.7 to 2.1. When higher concentrations of H2DIDS are applied (H2DIDS: hemoglobin = 5:0.5 mM), the Hill coefficient decreases further to 1.5 and the oxygen dissociation curve of hemoglobin is shifted slightly to the left of the control. Similar results were also obtained with DIDS instead of H2DIDS. SDS-PAGE shows that H2DIDS cross-links hemoglobin monomers mainly into dimers. Cross-linking is more effective under anaerobic conditions. With tritiated H2DIDS the larger part of the radioactivity is found in the dimer position of hemoglobin. Separation of the alpha and beta units of hemoglobin reacted with tritiated H2DIDS demonstrated a stoichiometry of 2.2 and 2.4 molecules H2DIDS per molecule alpha and beta unit hemoglobin, leading to about 8-9 H2DIDS molecules per native hemoglobin. The right shift produced in the hemoglobin oxygen dissociation curve and the cross-linking of monomers into dimers, especially under anaerobic condition, suggest that H2DIDS can also react with those amino groups of hemoglobin which are involved in 2,3-DPG binding. A comparison of H2DIDS, DIDS and 2,3-DPG at three different concentrations close to the hemoglobin concentration revealed a concentration dependent right shift in the oxygen dissociation curve with the order of potency 2,3-DPG greater than H2DIDS greater than DIDS. The Hill coefficients (n) at the three concentrations of 2,3-DPG demonstrated no changes, but H2DIDS and DIDS reduced in a concentration-dependent manner the cooperativity of hemoglobin. Again, H2DIDS is more potent than DIDS, especially at the low concentration. These anion-transport inhibitors provide novel approaches to the exploration of hemoglobin function.

2,3-Diphosphoglycerate↗
PubMed1985

Effects of vanadate, menadione and menadione analogs on the Ca2+-activated K+ channels in human red cells. Possible relations to membrane-bound oxidoreductase activity.

The modulation of the Ca2+- (or Pb2+-)activated K+ permeability in human erythrocytes by vanadate, menadione and chloro-substituted menadione analogs was investigated by measurements of K+ fluxes and single-channel currents. Vanadate and menadione stimulate the K+ permeability by increasing the probability of channel openings; the menadione analogs, on the other hand, inhibit the K+ permeability by increasing the probability of channel closings. The compounds used in these experiments also interact with oxidoreductases; it is demonstrated that menadione analogs in contrast to menadione strongly inhibit the membrane-bound dehydrogenase in the erythrocytes. Concentrations of Pb2+ above 10 mumol/l, but not of Ca2+, inhibit the enzyme activity as well as the K+ permeability. The parallel effects on dehydrogenase activity and the K+ channels suggest a direct relationship between these two systems in the membrane of erythrocytes.

Calcimycin↗
PubMed1985

Lead-induced activation and inhibition of potassium-selective channels in the human red blood cell.

The selective increase of net K+ permeability in human red cells brought about by either Ca2+ or lead was studied using a light scattering technique to measure net K+ fluxes in cell suspensions and the patch-clamp technique to study K+ transport in individual K+-selective channels of the red cell membrane. Using ultrapure solutions it was demonstrated that the effect of lead is neither the indirect consequence of a lead-induced increase of the accessibility of the receptor sites of the K+-selective channels to traces of Ca2+ that are present as contamination in analytical grade reagents nor to the release of Ca2+ from intracellular Ca2+ stores. It is further shown that in cell-free membrane patches low concentrations of lead (10 microM) in Suprapur solutions evoke the same single-channel events as added Ca2+ and that this activity can be inhibited by high concentrations of lead (100 microM), similar to the net KCl efflux measured by means of the light scattering technique. It is concluded, therefore, that both Ca2+ and lead independently activate the same K+-selective channels in the red cell membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗