Dissociation constant of the EnvZ/OmpR complex

Value 1.2 µM Range: ±0.17 µM
Organism Bacteria Escherichia coli
Reference Cai SJ, Inouye M. EnvZ-OmpR interaction and osmoregulation in Escherichia coli. J Biol Chem. 2002 Jul 5 277(27):24155-61 Table IPubMed ID11973328
Method His10-OmpR—His10-OmpR, purified as described previously, serves as a substrate for both kinase and phosphatase activities of EnvZ, similar to the wild-type OmpR protein (33). After His10-OmpR and EnvZc were mixed in the binding assay, both the EnvZ/His10-OmpR complex and free His10-OmpR were isolated from the mixture with Ni-NTA-agarose. The proteins bound to Ni-NTA-agarose were eluted and analyzed by SDSPAGE. The gels were stained with Coomassie Brilliant Blue, and the densities of the EnvZ and His10-OmpR bands were measured with a densitometer. The densities of EnvZ bands were normalized the relative density of EnvZc was 91% that of His10-OmpR when equimolar amounts of EnvZc and His10-OmpR were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. Also note that the densities of EnvZc and OmpR stained with Coomassie Brilliant Blue are linear in a range from 150 to 4800 ng (data not shown). The densities of the EnvZc and OmpR bands in the SDS-PAGE gel for the binding assay were within this range. Because OmpR molecules bind to EnvZc monomers stoichiometrically in a 1:1 ratio (2 OmpR molecules/1 EnvZc dimer), the percentage of the His10-OmpR bound to EnvZc can be estimated from the ratio of densities of EnvZc to His10-OmpR in the Coomassie Brilliant Blue-stained SDS-PAGE gel.
Comments The apparent Kd value of EnvZc and His10-OmpR interaction was 1.20±0.17µM under the conditions used. Assuming the cell volume is 1µM^3 (37), the cellular concentrations of OmpR and EnvZ were calculated to be 6 and 0.18µM, respectively. Therefore on the basis of the Kd value thus obtained, ~85% of EnvZ molecules in a cell are estimated to be occupied with OmpR. Calculation: the disscoiation constant of the reaction EnvZ/OmpR?EnvZ+OmpR is kd= ([EnvZ]×[OmpR])/[EnvZ/OmpR]. Using values above first to find [EnvZ/OmpR], the following calculation can be made: [EnvZ/OmpR]/([EnvZ/OmpR]+[EnvZ])=83.333%. See BNID 105109,105110
Entered by Uri M
ID 105111