Günter Fred Fuhrmann

Comparative studies of the structure and composition of the plasmalemma and the tonoplast in Saccharomyces cerevisiae

Abstract The two membranes, plasmalemma and tonoplast (Saccharomyces cerevisiae H 1022), are characterized ultrastructurally by their different texture in the corresponding freeze-fracture faces and their silver staining properties. Biochemical characterization with regard to proteins and lipids indicated that the ratio of protein to lipid is significantly higher in the plasmalemma as compared to the tonoplast. Moreover, a pronounced difference appears to exist for both the amount and the composition of total lipids, phospholipids and sterols. The protein patterns of the plasmalemma and the tonoplast reveal only minor differences, as judged by sodium dodecyl sulphate gel electrophoresis.

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.

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.

Kinetic analysis of glucose transport in wild-type and transporter-deficient Saccharomyces cerevisiae strains under glucose repression and derepression

Abstract Glucose transport in Saccharomyces cerevisiae is under regulatory control by glucose. After transition from glucose repression to derepression, the affinity of facilitated glucose transport into the cells increases from K m values around 7 mM to 1–2 mM. This change is accompanied by a significant increase of V m under these conditions. Published glucose-transport data obtained from the transporter-deficient single mutants snf3 , hxt2 and the double mutant snf3,hxt2 (Kruckeberg and Bisson, 1990) were evaluated by computer-assisted nonlinear regression analysis, which indicates that additional glucose transporters are encoded by other genes different from SNF3 and HXT2 . Regulation of glucose transport under glucose repression and derepression requires the presence of the SNF3 gene.

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.

Inhibition of glucose transport in Saccharomyces cerevisiae by uranyl ions

Abstract Transport of glucose into Saccharomyces cerevisiae cells occurs by two transport processes. The first transport is mathematically characterized by facilitated diffusion and the second by diffusion kinetics. Both transport processes are effectively blocked by uranyl ions. Facilitated diffusion of glucose demonstrates a mixed type of inhibition by uranyl ions; V m decreases and K m increases. The K i value for V m is 43.78 × 10 6 molecules uranyl per cell and the Hill slope 1.64. These values are comparable with the data on uranyl inhibition of glucose consumption by Rothstein, Frenkel and Larrabee (1948) with a K i value of 27.21 × 10 6 molecules uranyl per cell and a Hill slope of 2.51. By considering uranyl chemistry a dimeric complex of uranyl might act as a molecule similar to β- d -glucopyranose at the binding site of the carrier and by inhibition of the glucose translocation step. The effects on glucose diffusion seem to be more complex. At low concentrations of uranyl a significant inhibition occurs, whereas at higher uranyl concentrations a slight increase is observed. This dual behaviour of uranyl could be due to effects on two different diffusion pathways.

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.

Identification of 59 and 62 kDa plasma membrane proteins as putative glucose transporters in Saccharomyces cerevisiae

Abstract In this investigation two different glucose derivatives, the affinity label N-{O -(β- d -glucopyranosyl)ethylβromoacetamide (GEBrA) and the photoaffinity label 4-azi-4-deoxy- d -xylohexopyranose, were found to be effective inhibitors of glucose countertransport in plasma membrane vesicles prepared from Saccharomyces cerevisiae cells. Other common inhibitors of glucose transport for example in human red cells, cytochalasin B, the organic mercurial mersalyl, phlorizin and phloretin at low concentration were completely ineffective in inhibition. GEBrA and 4-azi-4-deoxy- d -xylohexopyranose were used radioactively to label membrane proteins of plasma membranes prepared from Saccharomyces cerevisiae cells. Both compounds labeled very distinctly two main integral membrane proteins at 59 and 62 kDa. These proteins are therefore the most probable candidates to represent the glucose carrier region in plasma membranes. By raising antibodies against these two membrane proteins at 59 and 62 kDa, the hypothesis that these proteins represent glucose carriers in the plasma membrane could further be supported. The polyvalent antibodies reacted only with plasma membrane proteins and not with proteins of other cellular membranes. Mainly the two proteins at 59 and 62 kDa were reactive. Reaction around 44 kDa is due to the deglycosylated forms of the double band. This SDS-PAGE region was also slightly labeled by the two glucose derivatives. Other minor labeled proteins with lower molecular masses are highly probably proteolytic products of the glucose carrier. The proteins labeled at 120 kDa by antibodies and glucose derivatives are most likely dimers of the double band at 59 and 62 kDa.

Dosis und Wirkung

In den vorangehenden Kapiteln wurden zahlreiche Wirkorte und Rezeptoren fur Sulfonamide sowie Zusammenhange zwischen der chemischen Struktur der Sulfonamide und ihrer Wirkung beschrieben. Es wurde auserdem die Vielseitigkeit der Wirkungen und Nebenwirkungen der Sulfonamide aufgezeigt. Im Falle der chemotherapeutischen Hauptwirkung waren die Bakterien das selektive Ziel ihres Angriffspunktes. Die Nebenwirkung der Carboanhydrasehemmung der Sulfonamide wurde exploriert, und systematische Sulfonamidsynthesen fuhrten schlieslich zu den Thiazid- und Schleifendiuretika. Die genauen Beobachtungen von Arzten am Krankenbett und von experimentierenden Forschern an Versuchstieren bei der Sulfonamidverabreichung waren der Ausgangspunkt fur die klinische Entwicklung von antidiabetischen Sulfonamiden und Thyreostatika.

Pharmako- und toxikodynamische Wirkung

Im vorangegangenen Teil der „Starthilfe Pharmakologie“ wurden Einflusse des Organismus auf Substanzen unter dem Oberbegriff Pharmako- und Toxikokinetik behandelt. Dabei wurden die Bewegungen der Substanzen im Organismus und die metabolischen Umsetzungen bis hin zu ihrer Ausscheidung verfolgt.