Effects of Divalent
Cations and Disulfide Bond Reducing Agents on Specific Binding of Growth
Hormone to Liver Membrane Receptors from Snakehead Fish (Ophiocephalus
argus, Cantor)
SUN Xun, ZHANG Xin-Na, ZHU Shang-Quan*
( Shanghai Institute of Biochemistry,the Chinese Academy of Sciences,
Shanghai 200031,China )
ZHENG Han-Qi
( Department of Biochemistry,Chinese University of Hong Kong,Hong
kong,China )
Abstract Divalent
cations, Ca2£«, Mg2£« and Mn2£« enhance the binding of bream
growth hormone (brGH) to snakehead fish liver membrane, and their optimum
concentration was found to be 8¡«12 mmol/L, at which Ca2£«, Mg2£« and Mn2£« could increase, respectively,
the specific binding to 230%, 180%, and 200%, compared with the binding in the
absence of ions. The Eadie-Scatchard plot was used for the dynamic analysis of
the Ca2£« binding site. A low affinity Ca2£« binding site was found
in the GH-receptor complex with Km=0.384 mmol/L, and the
affinity constant (Ka) was increased from 1.045¡Á109
L¡¤mol-1 to 1.295¡Á109 L¡¤mol-1 by the addition
of 10 mmol/L CaCl2. The effects of disulfide bond reducing agents,
DTT and ME, on 125I-brGH binding to growth hormone receptor (GHR) on
snakehead fish liver memebrane were also analyzed. The addition of 0.1¡«20 mmol/L DTT or 0.01%¡«1% ME to the radioreceptor
assay system caused a significant dose dependent increase in the specific binding
for 125I-brGH. In the presence of 0.8 mmol/L DTT or 0.08% ME, the
specific binding of 125I-brGH was increased from 10.2% to 15.5% and
13.2% respectively, and the affinity constant was also increased from 1.265¡Á109
L¡¤mol-1 to 2.185¡Á109 L¡¤mol-1 and 1.625¡Á109
L¡¤mol-1, respectively£» but no changes in the binding capacity
were observed. Further studies showed that the effects of reductants on the
specific binding of brGH were due in part to the ligand itself and in part to
GHR. In addition, it was observed that one of the three disulfide bonds of brGH
could be reduced by 0.8 mmol/L DTT.
Key words bream growth hormone£» growth hormone receptor£» receptor binding£» binding constant£» snakehead fish liver membrane£» divalent cation£» disulfide bond reducing agent
*Corresponding author£º Tel, 86-21-64374430-283£» Fax, 86-21-64338357£» e-mail, [email protected]