| Range |
250-300 Copies/cell
|
| Organism |
Bacteria Escherichia coli |
| Reference |
Farrell CM, Grossman AD, Sauer RT. Cytoplasmic degradation of ssrA-tagged proteins. Mol Microbiol. 2005 Sep57(6):1750-61.PubMed ID16135238
|
| Method |
Researchers sought to determine the intracellular levels of cytoplasmic proteases and adaptors known to be involved in
degradation of ssrA-tagged substrates in vitro and to monitor how these levels might change with nutrient availability. For these studies, E. coli strain X90 was grown at 37°C
in Neidhart’s complete medium (Neidhardt et al., 1974), a
rich but de?ned broth, and samples were taken for analysis during exponential growth and early stationary phase.
Quantitative Western blotting was used to measure the
levels of ClpX, SspB, ClpA and ClpP. For each molecule,
puri?ed protein at different concentrations was used to
generate a standard curve. Comparison of the standard
intensities with those of the cellular protein in the extract
allowed calculation of the intracellular concentration. Intracellular levels of ClpS were determined by a competitive
ELISA assay instead of Western blotting because the
latter assay had high background caused by cross-reactivity. |
| Comments |
During exponential-phase growth, there was enough
intracellular ClpA to form an average of 40–50 hexamers
per cell (Fig. 1A). As cells entered early stationary phase,
ClpA levels increased to a level of roughly 150 hexamers
per cell (Fig. 1A). ClpP levels per cell also increased from
an average of about 100 ClpP14 molecules during exponential-phase growth to 250–300 ClpP14 molecules as
cells entered early stationary phase (Fig. 1A). In contrast,
intracellular levels of ClpX and SspB changed far less, rising only about 20% throughout the experiment. On
average, there was enough ClpX to form approximately
75–100 hexamers and enough SspB to form approximately 140–160 dimers per cell (Fig. 1B). ClpS levels also
remained relatively constant at 250–300 molecules per
cell throughout the experiment (Fig. 1B). Tetradecamer=An oligomer having fourteen subunits. |
| Entered by |
Uri M |
| ID |
105106 |