Exact(5)
Fluorescence time courses were converted into GDP concentrations and initial rates were determined by linear regression.
Initial velocity reactions were from 1 to 4 min, and GDP concentrations were varied as indicated.
In addition to a drop in the GEC, the changes in GMP and GDP concentrations also resulted in altered GTP/GDP and GTP/GMP ratios.
Initial velocity assays containing EF-Ts were assayed at reactions times between 15 and 75 s, and the concentration of EF-Ts was held constant at 0.01 μM, while GDP concentrations were varied as indicated.
The biosensor response was calibrated as described for the GTPase assay above, but using the calibration in the presence of 1 mM GTP for all data to calculate GDP concentrations from the fluorescence signal.
Similar(55)
(B ) RF3 band intensity relative to S1 and RF1 plotted vs GDP concentration.
GDP concentration was chosen according to previous studies and the concentrations of citrate and the CAG combination were selected after dose-response studies (supplementary Figure 1).
The intensity of the band corresponding to RF3 decreased gradually to zero with increasing GDP concentration, but the S1 and RF1 intensities remained unaltered.
ATR ParM shows a fast fluorescence response (<200 ms response time) allowing real-time measurements of GDP concentration changes on the time scale of seconds.
When the concentration of extra GDP was increased, the RF3 band decreased gradually and virtually disappeared at 200 μM extra GDP concentration, while the RF1 band remained at 1 1 stoichiometry with S1.
However, in the absence of RF1 or at elevated GDP concentration, RF3 did not form a stable complex with the ribosome, in line with previous conclusions based on less direct experimental evidence (Zavialov et al., 2001, 2002).
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