Trehalose-mediated inhibition of the plasma membrane H+-ATPase from Kluyveromyces lactis: dependence on viscosity and temperature

Abstract

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 Kramer's 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.

FIG. 1.

Trehalose-mediated inhibition of the plasma membrane H⁺-ATPase. Initial rates of ATP hydrolysis were measured at 20°C as described in Materials and Methods. The reaction mixture contained 10 mM PIPES (pH 7.0), 80 mM KCl, 5 mM sodium azide, 5 mM phosphoenolpyruvate, 200 μM NADH, 12.5 IU of pyruvate kinase, 10.45 IU of lactic dehydrogenase, 5 mM MgCl₂, and the indicatedconcentrations of ATP; the final volume was 2 ml. The H⁺-ATPase (4.3 μg of protein in 4 μl) was added to start the reaction. (A) Plot of ATP hydrolysis versus ATP concentration at the following molar concentrations of trehalose: 0 (○), 0.2 (•), 0.4 (□), 0.5 (▪), 0.6 (▵), and 0.8 (▴). The points are the means of three independent experiments. In all cases, the standard deviation was less than 5%. The lines are the result of the best fit to the Hill equation (equation 2) by nonlinear regression. (B) Lineweaver-Burk plot of the data in panel A; the lines were obtained by linear regression of the data. (C) Replots of the slope (○) and the 1/v axis intercept (•) in panel B. The lines are the best fit to the equation y = a + b·exp(c·x), where a is the ordinate, b and c are constants, and x is the trehalose concentration.

FIG. 2.

Temperature effects on the kinetics of the plasma membrane H⁺-ATPase. Experimental conditions were as described for Fig. 1. The assay temperatures were 20°C (○), 25°C (•), 30°C (□), 35°C (▪), and 40°C (▵). The points are the means of three independent experiments. The standard deviations were less than 5% of the values. The lines are the result of the best fit to the Hill equation by nonlinear regression (equation 2).

FIG. 3.

Arrhenius plot for the plasma membrane H⁺-ATPase in the presence of different concentrations of trehalose. The points are the calculated V_max at each trehalose concentration and temperature. Trehalose molar concentrations were 0 (○), 0.2 (•), 0.4 (□), 0.5 (▪), 0.6 (▵), and 0.8 (▴). The lines were obtained by linear regression. (Inset) Plot of the energy of activation (E_a) versus trehalose concentration. E_a values were calculated from the line slopes as described by the Arrhenius equation (equation 3), and the line was the result of the fitting as described for Fig. 1C.

FIG. 4.

Effects of temperature on the viscosity of trehalose solutions. Each trehalose solution was prepared, poured into the viscometer, and degassed. Then the viscometer was immersed in a water jacket connected to a circulating water bath (Polyscience 9000) at the indicated temperature. Temperature in the viscometer was allowed to equilibrate 10 min before the falling times were read. The reaction mixture contained 10 mM PIPES, pH 7.0, and the following molar concentrations of trehalose: 0 (○), 0.2 (•), 0.4 (□), 0.5 (▪), 0.6 (▵), and 0.8 (▴).

FIG. 5.

Effects of medium viscosity on the V_max displayed by the H⁺-ATPase. Experiments were performed at different temperatures. The relative V_max (V_max0/V_max) was plotted against the relative viscosity (η/η₀). η₀ is the viscosity of the solution in the absence of trehalose, and V_max0 is the V_max in the absence of trehalose. Temperatures were 20°C (○), 25°C (•), 30°C (□), 35°C (▪), and 40°C (▵). The lines are the result of linear regressions of the data.

FIG. 6.

Glycerol-mediated inhibition of the plasma membrane H⁺-ATPase. Experimental conditions were as described for Fig. 1. Glycerol molar concentrations were 0 (○), 1 (•), 2 (□), 3 (▪), and 3.5 (▵). The lines are the result of fitting the data to the Hill equation (equation 2) by nonlinear regression.

FIG. 7.

Correlation between viscosity and H⁺-ATPase inhibition by trehalose, sucrose, or glycerol. Viscosity measurements were performed as described for Fig. 4 at 20°C. Enzyme activity was measured as described for Fig. 1. Solutes were trehalose (○), sucrose (•), or glycerol (□). (A) Plot of viscosity (η) versus mass fraction (c). The solid lines were the result of fitting the data to the Mooney equation (equation 4) by nonlinear regression. (B) Plot of the relative V_max (V_max0/V_max) against the relative viscosity (η/η₀) in the presence of different viscosity-generating agents. The lines are linear regressions of the data.