Fig 1.
Activity of ADH (A), CS (B), LDH (C), and Luc (in black on panel D).
The activity of each enzyme was recorded at the specific wavelength for each reaction. The different data points on panels (A), (B) and (C) represent the raw data readout from each time-point during the deactivation by high temperature. The lines represent the slopes calculated for the initial linear phase of each reaction. Due to the necessary mixing step in our protocol we miss the first few seconds of the reaction, thus we extrapolated the slopes to time zero of each reaction. The observed activity of the enzymes drop as shown by the decreasing slopes as a function of time at high temperature. (D) The deactivation of each enzyme was shown as % relative remaining activity: ADH (red), CS (blue), LDH (green), and Luc (black). The data for each enzyme was normalized to the activity of the non-stressed enzyme. The data was plotted as mean and 95% CI.
Table 1.
Loss of activity of each enzyme as a function of time at high temperature.
Fig 2.
Hsp70 (A), Hsp90 (B), and GroEL (C) protect citrate synthase against temperature deactivation.
The activity of the enzyme after temperature stress alone (CS AS) or in the presence of different ratios of the respective chaperone (and excess ATP where noted) was measured as described in the Materials and Methods section, and plotted on each panel. The bars represent the mean of the parallel measurements; the error bars indicate the 95% confidence intervals of each measurement. The data was normalized to the activity of the enzyme alone before it was subjected to temperature stress (CS BS). One-way ANOVA analysis was performed in each case to assess the significance of the differences of the protection effect of each chaperone compared to the CS AS. Panel D shows examples of the concentrations of chaperones used in previous experiments to assess protective activity.
Fig 3.
Enzymatic activity of CS is protected by ERD10 against temperature deactivation.
The activity of CS was recorded before (A) and after stress (B) of 44°C for 40min in the presence (■) and absence (•) of different concentration of ERD10 as described in the Material and Methods section. Activity is presented as percentage of the activity of the non-stressed CS without the addition of ERD10. Concentration of CS is constant at 4.5nM. The symbols represent the mean, and the error flags—the 95%CI.
Fig 4.
Effect of E. coli total protein extract on the deactivation of CS by high temperature.
The activity of citrate synthase was compared before and after temperature stress, between samples with and without the addition of total protein extract from cells grown at 37°C or 42°C as indicated in the grid. The addition of either type of extract significantly protects the activity of CS (shown by t-test). A slight, but not statistically significant, difference was observed between the protective effects of various cell extracts. The height of the bars represents the mean of at least 6 technical repeats, the error flags– 95% CI. The significance of the differences was assessed via an one-way ANOVA.