Salvador Uribe

Trehalose-enzyme interactions result in structure stabilization and activity inhibition. The role of viscosity

Stress resistance is essential for survival. The mechanisms of molecule stabilization during stress are of interest for biotechnology, where many enzymes and other biomolecules are increasingly used at high temperatures and/or salt concentrations. Diverse organisms, exhibit rapid synthesis and accumulation of the disaccharide trehalose in response to stress. Trehalose is also rapidly hydrolyzed as soon as stress ends. In isolated enzymes, trehalose stabilizes both, structure and activity. In contrast, at optimal assay conditions, trehalose inhibits enzyme activity. A general mechanism underlying the trehalose effects observed at all temperatures probably is the trehalose-mediated increase in solution viscosity that leads to protein domain motion inhibition. This may be analyzed using Kramers theory. The role of viscosity in the effects of trehalose is analyzed in examples from the literature and in studies on the plasma membrane H+-ATPase from Kluyveromyces lactis. In the cell, it may be proposed that the large concentration of trehalose reached during stress stabilizes structures through viscosity. However, once stress ends trehalose has to be rapidly hydrolyzed in order to avoid the viscosity-mediated inhibition of enzymes.

Measuring Solution Viscosity and its Effect on Enzyme Activity.

In proteins, some processes require conformational changes involving structural domain diffusion. Among these processes are protein folding, unfolding and enzyme catalysis. During catalysis some enzymes undergo large conformational changes as they progress through the catalytic cycle. According to Kramers theory, solvent viscosity results in friction against proteins in solution, and this should result in decreased motion, inhibiting catalysis in motile enzymes. Solution viscosity was increased by adding increasing concentrations of glycerol, sucrose and trehalose, resulting in a decrease in the reaction rate of the H+-ATPase from the plasma membrane ofKluyveromyces lactis. A direct correlation was found between viscosity (η) and the inhibition of the maximum rate of catalysis (Vmax). The protocol used to measure viscosity by means of a falling ball type viscometer is described, together with the determination of enzyme kinetics and the application of Kramers’ equation to evaluate the effect of viscosity on the rate of ATP hydrolysis by the H+-ATPase.

Interactions of calcium with yeast mitochondria

The interactions of Ca2+ with mitochondria from Saccharomyces cerevisiae were explored. Mitochondria were loaded with the metallochromic dye Fluo-3 to measure the concentration of free calcium in the matrix. Addition of EGTA or Ca2+ led to fluctuations in mitochondrial free calcium between 120 and 400 nM. Ca2+ variations were slower at 4 degrees C than at 25 degrees C or in the presence of phosphate instead of acetate. The net uptake of 45Ca2+ was higher with phosphate than with acetate. The optimum pH for Ca2+ uptake was 6.8. Ruthenium red did not affect the uptake of Ca2+. Addition of antimycin-A or uncouplers led to a small and transient release of Ca2+. Addition of EGTA or the monovalent cations Na+ or K+ resulted in higher release of Ca2+. Site I but not site II dependent O2 consumption was partially inhibited by EGTA. The effect of Ca2+ on NADH oxidation is similar to results reported with enzymes from mammalian sources which use NADH, such as the pyruvate, isocitrate and oxoglutarate dehydrogenases.

Control of mitochondrial matrix calcium: Studies using fluo-3 as a fluorescent calcium indicator

Fluo-3, a fluorescent Ca2+ indicator, is sequestered by isolated rat liver mitochondria and is an effective probe for evaluating the concentration and kinetics of change of mitochondrial matrix ionized calcium ([Ca2+]m) under a variety of conditions. At the wavelengths employed, there is no significant interference by auto-fluorescence. There is an insignificant release of the indicator over four hours and the loading and presence of fluo-3 has no effect on respiratory rate or oxidative phosphorylation. The [Ca2+]m steady state can be altered by the assay conditions, i.e. the presence of extra-mitochondrial Ca2+, Mg2+ phosphate and respiratory inhibitors. The total matrix ionized calcium represents a small percent (less than 0.01%) of the total mitochondrial calcium.

In Saccharomyces cerevisiae, Cations Control the Fate of the Energy Derived From Oxidative Metabolism Through the Opening and Closing of the Yeast Mitochondrial Unselective Channel

The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca2+ or Mg2+) or organic (hexyl or octyl-guanidine) cations was measured. The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (Δψ), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC. Addition of 0.4 mM PO4 did not close the YMUC, although it did enhance the sensitivity to Ca2+ (I50 decreased from 50 to 0.3 mM) and Mg2+ (I50 decreased from 5 to 0.83 mM Mg2+). The Ca2+ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell. Nonetheless, Mg2+, Ca2+, and PO4 exhibited additive effects. These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile. Octyl-guanidine (OG-I50 7.5 μM) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail. The PO4-dependent, Ca2+/Mg2+-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.

Toxicity of allelopathic monoterpene suspensions on yeast dependence on droplet size.

The toxic effects of the allelopathic nonsubstituted monoterpenes β-pinene and limonene on yeast,Saccharomyces cerevisiae, were proportional to the size of the monoterpene droplets in suspension. Both the toxic effects and the size of the droplets in suspension were decreased by adding different solvents with the monoterpene as follows: dimethylsulfoxide – dimethylformamide ≫ ethanol > dioxane. Oxygen consumption was inhibited about 80% by 1 mM β-pinene added in dimethylsulfoxide but less than 10% when β-pinene was added in dioxane. Parallel decreases in droplet size and toxic effects of either monoterpene were also induced by hydrating the monoterpene-dimethylformamide or monoterpene-dimethylsulfoxide before addition to yeast. Molecular aggregation may be a mechanism to potentiate the allelopathic properties of monoterpenes when these associate with diverse soil components.

Interactions of arsenate, sulfate and phosphate with yeast mitochondria.

In the presence of K(+), addition of ATP or ethanol to yeast mitochondria triggers the depletion of the transmembrane potential (DeltaPsi) and this is prevented by millimolar concentrations of phosphate (PO(4)). Different monovalent and polyvalent anions were tested for their protective effects on mitochondria from Saccharomyces cerevisiae. Only arsenate (AsO(4)) and sulfate (SO(4)) were as efficient as PO(4) to protect mitochondria against the K(+) mediated swelling, depletion of the DeltaPsi, and decrease in the ratio of uncoupled state to state 4 respiration rates. Protection by PO(4), SO(4) or AsO(4) was inhibited by mersalyl, suggesting that these anions interact with a site located in the matrix side. In addition, the effects of SO(4) and AsO(4) on the F(1)F(0)-ATPase were tested: both SO(4) and AsO(4) inhibited the synthesis of ATP following competitive kinetics against PO(4) and non-competitive kinetics against ADP. The mersalyl sensitive uptake of (32)PO(4) was not inhibited by SO(4) or AsO(4), suggesting that the synthesis of ATP was inhibited at the F(1)F(0)-ATPase. The hydrolysis of ATP was not inhibited, only a stimulation was observed when AsO(4) or sulfite (SO(3)) were added. It is suggested that the structure and charge similarities of PO(4), AsO(4) and SO(4) result in undiscriminated binding to at least two sites located in the mitochondrial matrix: at one site, occupation by any of these three anions results in protection against uncoupling by K(+); at the second site, in the F(1)F(0)-ATPase, AsO(4) and SO(4) compete for binding against PO(4) leading to inhibition of the synthesis of ATP.

Thermal inactivation of the plasma membrane H+-ATPase from Kluyveromyces lactis. Protection by trehalose.

The activity of the isolated plasma membrane H+-ATPase from Kluyveromyces lactis was measured during incubation at 35-45 degrees C and in the absence or in the presence of 0-0.6 M trehalose. As the temperature of incubation was raised from 35 to 45 degrees C, increasing enzyme inactivation rates were observed. Thermal inactivation kinetics of the H+-ATPase were biphasic exhibiting a first rapid phase and then a second slow phase. The transition from the native state occurred through a temperature-mediated increase in the inactivation rate constants of both phases. A model is proposed where the native H+-ATPase yields a partially active intermediary during the first phase of inactivation and then the intermediary is slowly converted into a totally inactive enzyme in the second phase. At each of these temperatures trehalose protected the enzymatic activity in a concentration dependent manner. Full protection was observed at 0.6 M trehalose in the range of 35-40 degrees C. Whereas, at 42 and 45 degrees C, the trehalose-mediated thermoprotection of the H+-ATPase was only partial. Trehalose stabilized the enzyme mainly by preventing the temperature dependent increase of the first and second inactivation rate constants.

Trehalose-Mediated Inhibition of the Plasma Membrane H+-ATPase from Kluyveromyces lactis: Dependence on Viscosity and Temperature

The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures. At 20 degrees C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V(max) and increased S(0.5) (substrate concentration when initial velocity equals 0.5 V(max)), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient V(max)/S(0.5) decreased from 5.76 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the absence of trehalose to 1.63 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the presence of 0.8 M trehalose. The decrease in V(max) was linearly dependent on solution viscosity (eta), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramers theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40 degrees C resulted in an exponential increase in V(max) and a decrease in enzyme cooperativity (n), while S(0.5) was not modified. As temperature increased, the effect of trehalose on V(max) decreased to become negligible at 40 degrees C, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in S(0.5) was similar at all temperatures tested, and thus, trehalose effects on V(max)/S(0.5) were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.

Multiple interactions of ethidium bromide with yeast cells

Abstract Experiments were carried out to determine the relationship between different energy states of the yeast cell and the uptake of ethidium bromide (EB). By varying the substrate, oxygenation, and by the use of uncouplers or respiratory inhibitors, it is possible to have energization or not of the whole cell, and also to deenergize specifically the mitochondria. The energy state of the whole cell can be determined by several means. With this system, three kinds of interactions of EB with the cell can be detected. The first one is a binding to the cell that does not seem to require energy. A second interaction is represented by the uptake of the dye into the cell, which does require energy, and is accompanied by an increase of the fluorescence of EB. The third interaction that can be monitored seems to be the uptake or binding of the dye by the mitochondria of the yeast cell; it requires specifically of the energization of this organelle, and manifests itself as a quenching of the fluorescence. The results are consistent with the hypothesis that the selectivity of EB for mitochondrial DNA can be partially explained by the ability of this organelle to concentrate the dye.