TY - JOUR
T1 - Determination of the dissociation constant of valine from acetohydroxy acid synthase by equilibrium partition in an aqueous two-phase system
AU - Engel, S.
AU - Vyazmensky, M.
AU - Barak, Z.
AU - Chipman, D. M.
AU - Merchuk, J. C.
N1 - Funding Information:
The help of Paulina Wainer in the work is gratefully acknowledged. This work was partially supported by grant 243/98 from the Israel Science Foundation.
PY - 2000/6/23
Y1 - 2000/6/23
N2 - An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, K(dis,) of the Escherichia coli isoenzyme AHAS III regulatory subunit, IlvH protein, from the feedback inhibitor valine. The amounts of the bound and free radioactive valine in the system were determined. A Scatchard plot of the data revealed a 1:1 valine-protein binding ratio and K(dis) of 133±14 μM. The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding. This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method. (C) 2000 Elsevier Science B.V.
AB - An aqueous polyethylene glycol/salt two-phase system was used to estimate the dissociation constant, K(dis,) of the Escherichia coli isoenzyme AHAS III regulatory subunit, IlvH protein, from the feedback inhibitor valine. The amounts of the bound and free radioactive valine in the system were determined. A Scatchard plot of the data revealed a 1:1 valine-protein binding ratio and K(dis) of 133±14 μM. The protein did not bind leucine, and the ilvH protein isolated from a valine resistant mutant showed no valine binding. This method is very simple, rapid and requires only a small amounts of protein compared to the presently used equilibrium dialysis method. (C) 2000 Elsevier Science B.V.
KW - Acetohydroxy acid synthase
KW - Aqueous two-phase systems
KW - Dissociation constant
KW - Partition coefficient
KW - Valine
UR - http://www.scopus.com/inward/record.url?scp=0034705787&partnerID=8YFLogxK
U2 - 10.1016/S0378-4347(00)00050-5
DO - 10.1016/S0378-4347(00)00050-5
M3 - Article
AN - SCOPUS:0034705787
SN - 1387-2273
VL - 743
SP - 225
EP - 229
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
IS - 1-2
ER -